Bicyclic signal transduction inhibitors, compositions containing them &amp; uses thereof

ABSTRACT

This invention concerns compounds for inhibiting intracellular signal transduction, especially intracellular signal transduction mediated by one or more molecular interactions involving a phosphotyrosine-containing protein. This invention also relates to pharmaceutical compositions containing the compounds and prophylactic and therapeutic methods involving pharmaceutical and veterinary administration of the compounds. The compounds are of the formula                    
     as defined herein.

RELATED APPLICATIONS

This application is a divisional of U.S. Ser. No. 09/438,601 (filed Nov.12, 1999), abn., which is a continuation in part of U.S. Ser. No.60/108,106 (filed Nov. 12, 1998), the full contents of both of which arehereby incorporated herein by reference.

FIELD OF THE INVENTION

This invention concerns a new class of compounds which have a broadrange of useful biological and pharmacological activities. Inparticular, these compounds are useful for inhibiting intracellularsignal transduction, especially intracellular signal transductionmediated by one or more molecular interactions involving aphosphotyrosine-containing protein. This invention also relates topharmaceutical compositions containing the compounds and prophylacticand therapeutic methods involving pharmaceutical and veterinaryadministration of the compounds.

BACKGROUND OF THE INVENTION

Cellular signal transduction, i.e., the series of events leading fromextracellular events to intracellular sequelae, is an aspect of cellularfunction in both normal and disease states. Numerous proteins thatfunction as signal transducing molecules have been identified, includingreceptor and non-receptor tyrosine kinases, phosphatases and othermolecules with enzymatic or regulatory activities. These moleculesgenerally demonstrate the capacity to associate specifically with otherproteins to form a signaling complex that can alter cell activity.

Signaling proteins often contain domain(s) of conserved sequence whichconstitute catalytic domains such as kinase or phosphatase domains, orserve as non-catalytic modules that direct protein:protein or otherinter- or intramolecular interactions during signal transduction. Suchdomains include among others, Src homology 2 (“SH2”) and phosphotyrosineinteraction (“PI”) domains. SH2 and PI domains recognize, i.e., bind to,proteins containing characteristic peptide sequences which include oneor more phosphorylated tyrosine (“pTyr”) residues. Significantinformation related to such domains, proteins containing them, theproduction of proteins containing such domains (including proteinfragments and fusion proteins), the characteristic peptide sequenceswhich they recognize and the biological and/or clinical role played bythe interactions of such proteins has been described in the scientificliterature. See e.g. U.S. Pat. No. 5,667,980, PCT/US97/02635(“Cell-Based Assay”) and WO 97/39326 (“In Vitro FluorescencePolarization Assay”) and references cited therein for additionalbackground information on SH2 and PI domains, inhibition ofintermolecular interactions mediated by such domains, assays and relatedtopics.

The protein domains of the tyrosine kinase, Src, gave rise to the “Srchomology” (“SH”) nomenclature and illustrate this class of proteins. Atleast nine members of the Src family of tyrosine kinases have beenidentified to date in vertebrates including Src (alternatively known asc-src and pp60c-src), Fyn, Yes, Lyn, Hck, Fgr, Blk and Yrk. Sequenceanalysis of the Src tyrosine kinases reveals that each family membercontains an N-terminal membrane anchor, a poorly conserved “unique”region of 40-70 amino acids, a Src homology 3 (SH3) domain of aboutsixty amino acids capable of protein-protein interactions withproline-rich sequences and a Src homology 2 (SH2) domain comprisingabout 100 amino acid residues which mediates binding of the Src familymember of phosphotyrosine-(pTyr) containing peptides and proteins(reviewed in Superti-Furga, FEBS Lett. 369:62-66 (1995). Several cognatephosphoproteins known to bind the Src SH2 domain include middle Tantigen, PDGF receptor, EGF receptor, and focal adhesion kinase (FAK).See Courtneidge et al, J. Virol. 65:3301-3308 (1991); Moi et al. EMBO J.12:2257-2264 (1993); Luttrell et al. Proc. Natl. Acad. Sci. USA 91:83-87(1994); and Xing et al, Mol. Biol. Cell 5:413-421 (1994). For additionalinformation on other SH2 domains (including, e.g., ZAP-70, Syk, Shc,Tsk, Btk, VAV, Grb2, Crk, STATs) and PI domain-containing proteins, seeWO 97/39326 and references cited therein.

The molecular structure of several SH2 domains has been solved and, inparticular, the molecular structure of certain SH2 domains in complexwith a phosphotyrosine-containing peptide or peptide analog has beenelucidated. See Waksman et al, Cell 72:779-790 (1993); Xu et al.Biochemistry 34:2107-2121 (1995); Hatada et al, Nature 377(6544), 32-38(1995). Whereas the general consensus sequence of Src family SH2-bindingpeptides, for example, comprises a pTyr-X-X-(Leu/Ile) motif, SH2 domainbinding specificity is thought to be influenced significantly by thespecific amino acids located carboxy-terminal to the pTyr residue. Forexample, the pp60c-src, Fyn, Lck and Fgr SH2 domains prefer the sequencepTyr-GluGlu-Ile. See Songyang et al, Cell 72:767-778 (1993).Crystallographic data concerning pp60c-src SH2 in complex with syntheticpeptides has revealed, in particular, two important binding determinantsfor binding to phosphotyrosine-containing proteins or peptides: thephosphotyrosine binding site which is electropositive in nature suchthat phosphotyrosine binding is stabilized and the lipophilic bindingsite which stabilizes binding of pTyr+3 residues within thephosphotyrosine-containing peptides via hydrophobic contacts. Reviewedby Brown and Cooper, Biochim. Biophys. Acta 1287 (2-3):121-149 (1996).

Structural studies of phosphotyrosine-containing peptides complexed withisolated SH2 domains has provided information regarding the molecularinteractions of peptide ligands with the SH2 domain peptidyl bindingsite. Recent attempts have been made to extrapolate these data to designnovel peptide ligands and peptidomimetic agonists of SH2-mediatedsignaling. For example, Plummer et al reported that incorporation ofC-terminal D-amino acid residues to tripeptide SH2 domain ligandsincreases affinity relative to their L-amino acid-containingcounterparts. See Plummer et al, Drug Design Discovery 13:75-81 (1996).Burke et al reported that hexapeptides containingdifluoro-(phosphonomethyl)phenylalanine bound SH2 domains with highrelative affinity compared to analogous pTyr peptides and were resistantto naturally-occurring cellular phosphatases. Studies of the pTyrresidue of peptide agonists of the Src SH2 domain have shown that thatphosphate ester is important for molecular recognition, and thatsignificant loss in binding occurs when it is replaced with sulfate,carboxylate, nitro, hydroxy or amino groups. See Gilmer et al, J BiolChem 269:31711-31719 (1994).

Many signaling pathways which play critical roles in disease processesare mediated by the binding of a phosphotyrosine-containing protein orprotein domain with an SH2 or other protein receptor for atyrosine-phosphorylated domain. Pharmaceutical agents which interferewith signaling mediated by such molecules, e.g., which interfere withthe formation or stability of such signaling complexes, may be used forprecise intervention in these complex biological processes in order totreat or prevent the diseases or pathological effects mediated by suchsignaling. Such interference may be achieved through a variety ofmechanisms, including competitive inhibition of aphosphotyrosine-containing ligand with its receptor (e.g., with anSH2-containing protein), inhibition of phosphorylation of the tyrosineresidue of such a ligand, inhibition of activation of a kinase whichcatalyzes the phosphorylation of a ligand in a signaling pathway, etc.

Compounds that can enter cells and block a signal transduction pathwayof interest, such as an SH2-mediated pathway, would be of great interestas reagents for biological research and for pharmaceutical andveterinary uses.

SUMMARY OF THE INVENTION

This invention concems compounds of Formula I, or pharmaceuticallyacceptable derivatives thereof:

in which

Y is

G is —O—, —S— or —NR—;

R⁶ comprises —OR, —APO₃RR′, —OPO₃RR′, —ASO₃R, —OSO₃R, —ACO₂R,—A-tetrazole, —A—N—(PO₃RR′)(PO₃RR′)′, —ASO₂NRR′, —ACOCF₃, —(C═O)J,—C(R)(J)(K) or —C(Z)(J)(K);

where each occurrence of A is independently a covalent bond, —G—M— or—(M)_(m)—;

each occurrence of M is an independently selected, substituted orunsubstituted, methylene moiety, and any M-M′ moiety may beelectronically saturated or unsaturated and/or may be part of a3-8-membered ring. Illustrative “M” moieties include —CH₂—, —CHF—,—CF₂—, —CHOH—, —CH(Me)—, etc.

Each n is independently 0, 1, 2, 3, 4 or 5 (in many embodiments n is 0,1 or 2);

each m is independently 0, 1 or 2;

J and K are independently selected from the group consisting of—APO₃RR′, —OPO₃RR′, —ASO₃R, —OSO₃R, —ACO₂R, —A-tetrazole, —ASO₂NRR′,

—(M)_(n)NRR′ and —(M)_(n)OR;

Z is a halogen (i.e., F, Cl, Br or I);

R⁷ and R⁸ are independently R, —CN, —NO₂, Z, J, —A(M)_(n)aliphatic,—G(M)_(n)aliphatic, —(M)_(n)COCF₃, —(M)_(n)OH, —(M)_(n)COOR,—A—(M)_(n)NRR′, —G—(M)_(q)NRR′, —(M)_(n)CHO, —A(M)_(n)N(R)(CO)R′,—A(M)_(n)N(R)(CO)GR′, —G(M)_(q)N(R)(CO)R′, —G—(M)_(q)N(R)(CO)G′R′,—A—(M)_(n)—CO—NRR′, or —G—(M)_(n)—CO—NRR′, where the aliphatic groupsmay be substituted or unsubstituted; or R⁷ is a covalent bond to an R⁴substituent of X forming an aliphatic, aryl or heterocyclic ring of 4 to8 atoms (including, for example a 5-membered nitrogen-containing ring ofan indole moiety). Each occurrence of R (unnumbered) represents hydrogenor an aliphatic, heteroaliphatic, aryl, heteroaryl, (aryl)aliphatic-, or(heteroaryl)aliphatic-moiety, each of which (other than hydrogen) may besubstituted or unsubstituted, e.g., with any of the various substituentslisted, illustrated or otherwise disclosed herein. While each occurrenceof “R” within a given compound is thus independently selected, wheremultiple R groups are depicted in the same figure or moiety, the variousR groups are generally marked R, R′, R″ and so on, as a reminder thatthey may be the same or different. (The same is true in the case ofnumbered “R” groups and other variables such as “m”, “n”, “M”, etc.where apostrophes are used for the same purpose. Note also that the n Mgroups in a “M_(n)” moiety may be the same or different from oneanother.)

q is an integer from 1 to 8, and in many embodiments is 1, 2 or 3;

X is: —(CR³R⁴)_(m)— or —NR⁴—;

R³ is hydrogen, R(CO)NR′—, RR′N(CO)NR″—, R′SO₂NR—, R′CSNR—, RR′NCSNR″—,RR′NSO₂NR″—, R′OCONR—, RR′N—, or

R⁴ is hydrogen, aliphatic (which may be branched, unbranched or cyclic),cycloaliphatic-(M)_(n)—, aryl-(M)_(n)—, heterocyclic-(M)_(n)—,RSO₂(M_(n))—, (CO₂R)(M)_(n)— or (RR′N—CO)(M)_(n), where the aliphatic,cycloaliphatic, aryl and heterocyclic groups are substituted orunsubstituted;

B is

where

E is M, G or one of the following:

p is 1, 2, 3 or 4;

R⁹, R¹⁰, R¹¹ and R¹² are independently —(M)_(n)Z, —(M)_(n)R, —(M)_(n)GR,—(M)_(n)WR or —(M)_(n)WGR, including, among others, moieties such as R,—OR, —SR, —CHO, —COR, —COOH (or amide, ester, carbamate, urea, oxime orcarbonate thereof, —NH₂ (or substituted amine, amide, urea, carbamate orguanidino derivative therof), halo, trihaloalkyl, cyano, —SO₂—CF₃,—OSO₂F, —OS(O)₂R, —SO₂—NHR, —NHSO₂R, sulfate, sulfonate, aryl andheteroaryl moieties. Alternatively, R¹⁰ and R¹¹ are covalently linkedtogether to form an aliphatic, hetercyclic or aryl fused ring, typicallyof 5-7 members. For example, in some embodiments, R¹⁰ and R¹¹ comprise—G—(M)_(n)—G′—, as illustrated by the following structure for B where,for the sake of example, each M is —CH₂— and n is 3:

where in some cases G is —O— and G′ is —S—, for example.

R¹⁴ is R (H is generally preferred); and,

U and W are independently —CO—, —CS—, —M—, —SO—, or —SO₂—:

a pharmaceutically acceptable derivative thereof.

Compounds of Formula I thus include compounds having the followingstructures:

and comprise a number of subgenera of particular interest.Representative subgenera are illustrated in the examples which follow.

One subgenus includes compounds in which at least one R⁴ moiety is H andat least one R³ moiety is either H or NH₂. Compounds of the latter sortinclude those in which X is

Also of interest are the subgenera of compounds in which the nitrogenatom of the moiety X is further elaborated, as depicted below:

where R⁵ comprises a substituted or unsubstituted, lower (i.e.,containing 1-8 carbon atoms) aliphatic or alkoxyl group, or is asubstituted or unsubstituted —(M)_(n)-aryl or —(M)_(n)-heterocyclic(including e.g., substituted and unsubstituted phenyl or benzyl group,or a homolog and heterocyclic analog thereof, including e.g.,2-naphthyl, 3-indolyl, and 1-imidazolyl).

Such compounds are further illustrated by the subset thereof in which R⁵comprises —(M)_(n)CH₃, —(M)_(n)aryl, —(M)_(n)heterocyclic, —(M)_(n)CN,—(M)_(n)COOR, where n is 0, 1, 2, 3, 4, or 5. For instance, in some suchcompounds R⁵ is a substituted or unsubstituted methyl, ethyl, n-propyl,i-propyl, n-butyl, sec-butyl, t-butyl, n-pentyl, sec-pentyl, i-pentyl,cyclo pentyl, etc. or benzyl moiety. In other such compounds R⁵comprises —(CH₂)_(n)CH₃, —(CH₂)(CH₂)_(n)aryl,—(CH₂)(CH₂)_(n)heterocyclic, —(CH₂)(CH₂)_(n)CN or —(CH₂)(CH₂)_(n)COOR,where n again is 0, 1, 2, 3, 4, or 5. Examples of such compounds includethose in which R⁵ comprises —CH₂CN, —(CH₂)CO₂R, —(CH₂)₂CO₂R,—(CH₂)₃CO₂R, —(CH₂)₄CO₂R, where R is H, lower alkyl or benzyl.

In some embodiments of compounds of the structure

R⁵ comprises —O—(M)_(n)CH₃, —O(M)_(n)aryl, —O(M)_(n)heterocyclic,—O(M)_(n)CN, or —O(M)_(n)COOR, where n is 0, 1, 2, 3, 4, or 5. Inspecific cases, R⁵ comprises —O(CH₂)_(n)CH₃, —O(CH₂)(CH₂)_(n)aryl,—O(CH₂)(CH₂)_(n)heterocyclic, —O(CH₂)(CH₂)_(n)CN, or—O(CH₂)(CH₂)_(n)COOR. In numerous cases, R⁵ comprises —O-(substituted orunsubstituted lower alkyl or benzyl.

Another subgenus of interest includes amides of the formula:

where R⁴ is hydrogen, substituted or unsubstituted aliphatic (which maybe branched, unbranched or cyclic), substituted or unsubstitutedaryl-(M)_(n)—, substituted or unsubstituted heterocyclic-(M)_(n)—, or(CO₂R)(M)_(n)—. Such compounds are illustrated by those in which R⁴ is—(M)_(n)(CO)OR, —(M)_(n)SO₂R, —(M)_(n)(CO)NRR′, or —(M)_(n)(tetrazole),including, for example, compounds in which R⁴ is —CH₂COOR, —CH₂SO₂R,—CH₂(CO)NRR′, or -tetrazole. Simple members of this subgenus are thosein which the R group(s) of R⁴ is (are independently) H, lower alkyl(e.g., methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tbutyl,etc.) or benzyl.

Another subgenus includes ureas of the formula:

where R¹, R², R⁴, R¹⁴, Y and m are defined as above. Thus, R⁴ may besimply H or may be a more complex R⁴ moiety such as are noted above.

Another subgenus includes amides of the formula:

In many examples of all of the foregoing compounds, one or more Rmoieties (R′, R″ etc) are H. Also, in many compounds of interest, R¹⁴ isH.

Compounds of Formula I, including, among others, compounds of thevarious subgenera described above, include those in which Y comprises

Such compounds in which R⁶ comprises —OR, —APO₃RR′, —OPO₃RR′, —ASO₃R,—OSO₃R, —ACO₂R, —A-tetrazole, —A—N—(PO₃RR′)(PO₃RR′)′, —ASO₂NRR′,—ACOCF₃, —C(R)(J)(K) or —C(Z)(J)(K) are of particular interest.Embodiments in which R⁷ and R⁸ are independently H, —CN, —NO₂, halogen,J, —A—(M)_(n)aliphatic, —G—(M)_(n)aliphatic, —(M)_(n)COCF₃, —(M)_(n)OR,—(M)_(n)COOR, —A—(M)_(n)NRR′, —G—(M)_(q)NRR′, —(M)_(n)CHO,—A—(M)_(n)N(R)(CO)R′, —G—(M)_(q)N(R)(CO)R′, —A—(M)_(n)—CO—NRR′, or—G—(M)_(n)—CO—NRR′, where the aliphatic groups may be substituted orunsubstituted; or R⁷ is a covalent bond to an R⁴ substituent of X toform a ring of 4 to 8 atoms are also of particular interest (includingamong others, those compounds in which the R groups of R⁶ and/or of R⁷and R⁸ are H). This set of compounds is illustrated by those in which R⁶comprises —OR, —APO₃RR′, —OPO₃RR′, —ACO₂R, —ACOCF₃ or —C(R)(J)(K); Acomprises —M_(m)— (e.g., —CH₂—, —CF₂—, —CHF—, —CHOH—, —CH₂CF₂—, etc),—GM— (e.g. —OCH₂—) or a covalent bond; each R and R′ is H, orsubstituted or unsubstituted lower alkyl or substituted or unsubstitutedbenzyl; and, R⁷ and R⁸ are independently H, J, —A—(M)_(n)substituted orunsubstituted aliphatic, —(M)_(n)COCF₃, —(M)_(n)OH, —(M)_(n)COOR,—A—(M)_(n)NRR′, —(M)_(n)CHO, —A—(M)_(n)N(R)(CO)R′ or —A—(M)_(n)—CO—NRR′.For example, in some such cases, R⁶ comprises —OH, —PO₃RR′, OPO₃RR′,—CH₂PO₃RR′, —CF₂PO₃RR, —OCH₂CO₂R, —NHCH₂CO₂R, —CH₂CO₂R, —CF₂CO₂R,—N(PO₃RR′)(PO₃RR′)′, —CH₂SO₃R, —CF₂SO₃R, —CH₂COCF₃, —CF₂COCF₃,—CH(PO₃RR′)₂, —CH(OH)(PO₃RR′), —CH(NH₂)(PO₃RR′), —CH(CO₂R)₂, —CF(CO₂R)₂,—CH(PO₃RR′)(CO₂R″), —CH(PO₃RR′)(SO₃R″), —CH(PO₃RR′)(SO₂NH₂),—CH(SO₂NH₂)₂, or —CH(SO₃RR′)₂. In some such compounds, one or more of R,R′ and R″ in the —PO₃RR′, —OPO₃RR′, —CH₂PO₃RR′, —CF₂PO₃RR′, —OCH₂CO₂R,—NHCH₂CO₂R, —CH₂CO₂R, —CF₂CO₂R, —CF₂CO₂R, —N(PO₃RR′)(PO₃RR′)∝, —CH₂SO₃R,—CF₂SO₃R, —CH₂COCF₃, —CF₂COCF₃, —CH(PO₃RR′)₂, —CH(OH)(PO₃RR′),—CH(NH₂)(PO₃RR′), —CH(CO₂R)₂, —CF(CO₂R)₂, —CH(PO₃RR′)(CO₂R″),—CH(PO₃RR′)(SO₃R″), —CH(PO₃RR′)(SO₂NH₂), —CH(SO₂NH₂)₂, or —CH(SO₃RR′)₂moiety is H. In others, one or more of those R groups is —(M)_(m)—CH₂Z,—(M)_(m)—CHZ₂, —(M)_(m)—CZ₃, —R¹⁵, —M—O—CO—OR¹⁵ or —M—O—R¹⁵, where Z ishalogen and R¹⁵ is substituted or unsubstituted lower aliphatic, aryl orheterocyclic. For example, in various embodiments, R¹⁵ is methyl, ethyl,n-propyl, i-propyl, n-butyl, isobutyl, t-butyl, n-amyl, sec-amyl, benzylor substituted benzyl, and M is CH₂, CHR (e.g. CHCH₃ etc.) and the like.Further illustrations include —CH₂—O—CO—OEt, —CH(Me)—O—CO—OEt,—CH₂—O—CO—t-butyl, etc.

In one subgenus of the foregoing compounds, R⁷ and R⁸ are both H. Inanother subgenus R⁷ is —N(M_(n)COOR)(M_(n)COOR)′, e.g., —N(CH₂CO₂R)₂. Inanother subgenus, R⁷ is J, —A—(M)_(n)(substituted or unsubstitutedaliphatic, aryl or heterocyclic), —(M)_(n)COCF₃, —(M)_(n)OH,—(M)_(n)COOR, —A—(M)_(n)NRR′, —(M)_(n)CHO, —A—(M)_(n)N(R)(CO)R′,—A—(M)_(n)—NRR′ or —A—(M)_(n)—CO—NRR′; and R⁸ is H. The latter subgenusis illustrated by compounds in which R⁷ is lower alkyl, lower alkenyl,—OH, —NH₂, —NO₂, —CN, —NHR, —NHCOR, —CHO, —CH₂CHO, —PO₃RR′, —OPO₃RR′,—CH₂PO₃RR′, —CF₂PO₃RR′, —OCH₂CO₂R, —NHCH₂CO₂R, —CH₂CO₂R, —CF₂CO₂R,—SO₃R, —CH₂SO₃R, —CF₂SO₃R, —COCF₃, —COCH₂F, —COCF₂H, —CF₂COCF₃ or—SO₂NH₂. In some such compounds, one or both of R and R′ in —PO₃RR′,—OPO₃RR′, —CH₂PO₃RR′, —CF₂PO₃RR′, —OCH₂CO₂R, —NHCH₂CO₂R, —CH₂CO₂R,—CF₂CO₂R, —SO₃R, —N(PO₃RR′)(PO₃RR′)′,—CH₂SO₃R, or —CF₂SO₃R is H. Inothers, one or more of those R groups is —(M)_(m)—CH₂Z, —(M)_(m)—CHZ₂,—(M)_(m)—CZ₃, —R¹⁵, —M—O—CO—OR¹⁵ or —M—O—CO—R¹⁵, where Z is halogen andR¹⁵ is substituted or unsubstituted lower aliphatic, aryl orheterocyclic. For example, in individual cases, R¹⁵ is methyl, ethyl,n-propyl, i-propyl, n-butyl, isobutyl, t-butyl, n-amyl, sec-amyl, benzylor substituted benzyl, and M is CH₂, CHR (e.g. CHCH₃ etc.) and the like.

In an illustrative subgenus, R⁶ comprises —APO₃RR′ (e.g., —OPO₃H₂) andR⁷ is —A—(M)_(n)substituted or unsubstituted aliphatic.

In another subgenus, R⁶ and R⁷ are independently selected from J and K.

In another subgenus, R⁶ is —C(R)(J)(K). Illustrative compounds of thissubgenus include those in which R⁶ is —CH(J)(K) and those in which R⁶ is—C(R)(PO₃R′R′)(K). The latter compounds are illustrated by embodimentsin which none, one, two or three of the R groups of the—C(R)(PO₃R′R′)(K) moiety are H.

As in previously mentioned cases, compounds of this invention whichcontain a moiety J, e.g., compounds of Formula I in which R⁶ is—C(R)(J)(K), include among others embodiments in which one or both of Rand R′ (e.g., of a —PO₃RR′ moiety) are R¹⁵, —(M)_(m)—CH₂Z,—(M)_(m)—CHZ₂, —(M)_(m)—CZ₃, —M—O—CO—OR¹⁵ or —M—O—CO—R¹⁵, where Z ishalogen and R¹⁵ is substituted or unsubstituted lower aliphatic, aryl orheterocyclic (e.g., methyl, ethyl, n-propyl, i-propyl, n-butyl,isobutyl, t-butyl, n-amyl, sec-amyl, benzyl or substituted benzyl), andM is CH₂, CHR (e.g. CHCH₃ etc.) and the like.

The compounds of Formula I, including the various subgenera andillustrative examples described above, all contain a bicyclic moiety, B,as that term is defined herein and as is illustrated by the followingformula:

Compounds of this invention include those in which each of R⁹, R¹⁰, R¹¹and R¹² is independently —(M)_(n)Z, —(M)_(n)R, —G(M)_(n)R, —(M)_(n)WR or—(M)_(n)W—GR. In certain embodiments, one or more of the R, R′ and R″groups of R⁹, R¹⁰, R¹¹, and R¹² comprise a halo, hydroxy, aliphatic,amino, amido or sulfonamido moiety. In some embodiments, one or more ofR⁹, R¹⁰, R¹¹, and R¹² is a substituted aliphatic moiety containing atleast one substituent selected from substituted or unsubstitutedcycloaliphatic, substituted or unsubstituted aryl, substituted orunsubstituted heteroaryl, —COR, —CO₂R, —CO—NRR′, and —OR. In someembodiments of particular interest, one or more of R⁹, R¹⁰, R¹¹, and R¹²comprises —(M)_(n)(cycloaliphatic), —(M)_(n)(substituted orunsubstituted aryl), —(M)_(n)(substituted or unsubstituted heteroaryl),—(M)_(n)CHO, —(M)_(n)CONH₂, —(M)_(n)CSNH₂, —(M)_(n)SONH₂,—(M)_(n)SO₂NRR′, —(M)_(n)OR, —(M)_(n)(lower aliphatic),—(M)_(n)—C(OR)R′R″, or —(M)_(n)—C═CRR′. For example, in some cases, oneor more of R⁹, R¹⁰, R¹¹, and R¹² comprise methyl, —(CH₂)_(q)R¹³ where qis 1-8 and R¹³ comprises methyl; i-propyl; i-butyl; t-butyl;cycloaliphatic; phenyl; substituted phenyl; naphthyl; substitutednaphthyl; a 5, 6 or 7-membered heterocyclic ring or a bicyclicheterocylic moiety. In some cases, R¹² comprises a formyl group on aring nitrogen. Possible substituents on the R, R′ and R″ groups include,among others, halo, hydroxy, alkyl, amino, amido and sulfonamidomoieties. Other potential substituents are as disclosed elsewhereherein, including in the numerous specific examples.

Other compounds of Formula I which are of particular interest includethose in which one or more of R⁹, R¹⁰, R¹¹, and R¹² comprises—G(M)_(n)(aliphatic), —G(M)_(n)(cycloaliphatic), —G(M)_(n)(substitutedor unsubstituted aryl), —G(M)_(n)(substituted or unsubstitutedheteroaryl), —G(M)_(n)CHO, —G(M)_(n)CONH₂, —G(M)_(n)CSNH₂,—G(M)_(n)SONH₂, —G(M)_(n)SO₂NRR′, —G(M)_(n)OR, —G(M)_(n)(loweraliphatic), —G(M)_(n)C(OR)R′R″, or —G(M)_(n)—C═CRR′, for instance asillustrated by cases in which —G(M)_(n) comprises —OCH₂—, —SCH₂— or—NRCH₂—.

Compounds of particular interest further include those in which one ormore of R⁹, R¹⁰, R¹¹, and R¹² comprises —(M)_(n)W—NH—R, e.g.,—(M)_(n)(CO)—NH—R, as illustrated by —(CH₂)_(m)CONH—R and —C(O)NR, forexample). In some compounds one or more of R⁹, R¹⁰, R¹¹, and R¹²comprises —O(M)_(m)(aliphatic).

In some cases R¹¹ and R¹² are both H, as illustrated by the followingstructure:

In illustrative embodiments, R⁵ comprises a substituted or unsubstitutedlower aliphatic moiety, R⁹ comprises —(M)_(n)W—NH—R″ and R¹⁰ comprises—O(M)_(m)(aliphatic). For example, in some such compounds, R⁹ comprises—CONH—R″ and R¹⁰ comprises —OM-cycloaliphatic or —OM-branched chainaliphatic. In other cases, R⁹ comprises —CH₂CONH—R and R¹⁰ comprises—OM-cycloaliphatic or —OM-branched chain aliphatic.

From the perspective of Y moieties, compounds of interest include thosecompounds of Formula I, including those of the various subgenera andexamples herein, in which Y comprises

particularly where R⁶ comprises —OR′, —APO₃R′R″, —ASO₃R′, —ACO₂R′,—ASO₂NR′R″, —ACOCF₃, or —C(R′)(J)(K); and, R⁷ is H, —CN, —NO₂, halogen,J, —A—(M)_(n)substituted or unsubstituted aliphatic, —(M)_(n)COCF₃,—(M)_(n)OH, —(M)_(n)COOR, —A—(M)_(n)NRR′, —(M)_(n)CHO,—A—(M)_(n)N(R)(CO)R′ or —A—(M)_(n)—CO—NRR′. For example, in some casesR⁶ comprises —PO₃RR′, —OPO₃RR′, —OSO₂NRR′, —(CH₂)PO₃RR′, —(CF₂)PO₃RR′ or—CRJK; and R⁷ comprises R (including among others, H, alkyl, alkenyl,etc.) —CN, amido, acylamino, J (e.g. —CO₂R), or —CHO. For example, insome cases, R⁶ comprises —OPO₃RR′ or —(CF₂)PO₃RR′ and R⁷ is H. In someembodiments one or more R groups (including R′, R″, etc) of R⁶ comprises—(M)_(m)—CH₂Z, —(M)_(m)—CHZ₂, —(M)_(m)—CZ₃, —R¹⁵, —M—O—CO—OR¹⁵ or—M—O—CO—R¹⁵, where Z is H or halogen and R¹⁵ is substituted orunsubstituted lower aliphatic, aryl or heterocyclic. For example, inindividual cases, R¹⁵ is methyl, ethyl, n-propyl, i-propyl, n-butyl,isobutyl, t-butyl, n-amyl, sec-amyl, benzyl or substituted benzyl, and Mis CH₂, CHR (e.g. CHCH₃ etc.) and the like.

Compounds of the structure

(as well as homologs in which p is 1 or 2) are of interest asintermediates in the preparation of compounds of this invention. Ofparticular interest are such compounds in which R⁹ and R¹⁰ areindependently halo, R, —OR, —SR, —NRR′, —COR, or —(M)_(n)W—NHR, and R¹¹and R¹² are as previously defined, including cases in which R¹¹ and R¹²are H.

Compounds of this invention which are of special interest include thosewhich bind to a given SH2 domain (or protein containing such SH2 domain)with a IC₅₀ value of less than 50 μM, preferably less than 20 μM, asdetermined by any scientifically valid method, in vitro or in vivo. SH2domains of current interest include those of a Src, Fyn, Lck, Yes, Blk,Lyn, Fgr, Hck, Yrk, ZAP-70, Syk, STAT or Abl protein.

Also of interest are pharmaceutical compositions comprising a compoundof this invention, or a pharmaceutically acceptable derivative thereof,and one or more pharmaceutically acceptable excipients.

Compounds of this invention (or a composition containing such acompound) can be administered to cells or to animals, preferably amammal in need thereof, as a method for inhibiting SH2-mediated signaltransduction therein. In particular cases, it will be advantageous tocarry out that method using a pharmaceutical composition containing acompound which specifically binds to an SH2 domain of Src, ZAP-70, Syk,or STAT 6. In other cases it will be advantageous to carry out thatmethod where the SH2-mediated signal transduction is mediated by a PDGFreceptor protein, EGF receptor protein, HER2/Neu receptor protein,fibroblast growth factor receptor protein, focal adhesion kinaseprotein, p130 protein, or p68 protein.

Cases in which a mammal may be in need of inhibition of SH2-mediatedsignaling include cases in which the mammal has a proliferative disease,cancer, restenosis, osteoporosis, inflammation, allergies, orcardiovascular disease. In such cases, administering a therapeuticallyeffective amount of the composition to the mammal, preferably to a humanpatient, will constitute treating or preventing the proliferativedisease, cancer, restenosis, osteoporosis, inflammation, allergicreaction, or cardiovascular disease in the recipient or a method forcausing immunosuppression in the recipient.

Generally preferred compounds of this invention include any of theforegoing compounds which yield an observable IC₅₀ value, when testedagainst an SH2 domain of interest and a pTyr-containing peptide ligand(or mimic thereof for that SH2 domain, of 50 μM or better, preferably 5μM or better, more preferably 1 μM or better, and even more preferably,500 nM or better, as determined by any scientifically valid measure,especially when the SH2 domain is from a Src, Fyn, Lck, Yes, Blk, Lyn,Fgr, Hck, Yrk, ZAP, Syk, STAT or Abl protein.

A pharmaceutical composition may be prepared containing a compound ofthis invention (including a pharmaceutically acceptable derivativethereof) together with one or more pharmaceutically acceptableexcipients.

A compound of this invention, preferably in the form of a pharmaceuticalcomposition, may be administered to a mammal in need thereof, preferablya human patient, as a method for inhibiting SH2-mediated signaltransduction in the recipient mammal. In some cases, the compound may beselected based on its ability to specifically bind to an SH2 domain,e.g. of Src, ZAP-70, Syk, or STAT 6, etc., or on its ability to inhibita signal transduction pathway mediated by an SH2 domain-containingprotein. Such use of an appropriately selected compound of thisinvention thus provides a method for inhibiting SH2-mediated signaltransduction which is mediated by a PDGF receptor protein, EGF receptorprotein, HER2/Neu receptor protein, fibroblast growth factor receptorprotein, focal adhesion kinase protein, p130 protein, or p68 protein.Use of a compound of this invention may be particularly advantageous incases in which the mammal has a proliferative disease, cancer,restenosis, osteoporosis, inflammation, allergies, or cardiovasculardisease. In such cases, administering to the patient a therapeuticallyeffective amount of a compound of this invention, preferably in the formof a pharmaceutical composition, provides a method for treating orpreventing a proliferative disease, cancer, restenosis, osteoporosis,inflammation, allergies, or cardiovascular disease in the patient.

DETAILED DESCRIPTION OF THE INVENTION

Compounds and Definitions

As mentioned above, this invention provides a novel class of compoundsuseful as inhibitors of signal transduction pathways mediated by theinteraction of protein receptors for phosphotyrosine-containingproteins, such as proteins containing one or more SH2 domains, withtheir phosphotyrosine-containing ligands. Compounds of this inventioncomprise those of Formula I, set forth above, and are illustrated inpart by the various classes, subgenera and subsets of compounds notedabove, and by the various subgenera and species disclosed elsewhereherein. The compound may be in the form of an individual enantiomer,diastereomer or geometric isomer, or may be in the form of a mixture ofstereoisomers.

Also included are pharmaceutically acceptable derivatives of theforegoing compounds, where the phrase “pharmaceutically acceptablederivative” denotes any pharmaceutically acceptable salt, ester, or saltof such ester, of such compound, or any other adduct or derivativewhich, upon administration to a patient, is capable of providing(directly or indirectly) a compound as otherwise described herein, or ametabolite or residue thereof, preferably one which is a signaltransduction inhibitor. Pharmaceutically acceptable derivatives thusinclude among others pro-drugs. A pro-drug is a derivative of acompound, usually with significantly reduced pharmacological activity,which contains an additional moiety which is susceptible to removal invivo yielding the parent molecule as the pharmacologically activespecies. An example of a pro-drug is an ester which is cleaved in vivoto yield a compound of interest. Pro-drugs of a variety of compounds,and materials and methods for derivatizing the parent compounds tocreate the pro-drugs, are known and may be adapted to the presentinvention.

The term “aliphatic” as used herein includes both saturated andunsaturated, straight chain (i.e., unbranched), branched, cyclic, orpolycyclic aliphatic hydrocarbons, which are optionally substituted withone or more functional groups. Unless otherwise specified, alkyl, otheraliphatic, alkoxy and acyl groups preferably contain 1-8, and in manycases 1-6, contiguous aliphatic carbon atoms. Illustrative aliphaticgroups thus include, for example, methyl, ethyl, n-propyl, isopropyl,cyclopropyl, —CH₂-cyclopropyl, allyl, n-butyl, sec-butyl, isobutyl,tert-butyl, cyclobutyl, —CH₂-cyclobutyl, n-pentyl, sec-pentyl,isopentyl, tert-pentyl, cyclopentyl, —CH₂-cyclopentyl, n-hexyl,sec-hexyl, cyclohexyl, —CH₂-cyclohexyl moieties and the like, whichagain, may bear one or more substituents.

Some examples of substituents of aliphatic (and other) moieties ofcompounds of this invention include: R, —OH, —OR, —SH, —SR, —CHO, ═O,—COR, —COOH (or amide, ester, carbamate, urea, oxime or carbonatethereof, —NH₂ (or substituted amine, amide, urea, carbamate or guanidinoderivative therof, halo, trihaloalkyl, cyano, —SO₂—CF₃, —OSO₂F,—OS(O)₂R, —SO₂—NHR, —NHSO₂R, sulfate, sulfonate, aryl and heteroarylmoieties. Aliphatic, heteraliphatic, aryl and heterocyclic substituentsmay themselves be substituted or unsubstituted (e.g. mono-, di- andtri-alkoxyphenyl; methylenedioxyphenyl or ethylenedioxyphenyl;halophenyl; or -phenyl-C(Me)₂—CH₂—O—CO—[C3-C6] alkyl or alkylamino).Additional examples of generally applicable substituents are illustratedby the specific embodiments shown in the Examples which follow.

The term “aliphatic” is thus intended to include alkyl, alkenyl,alkynyl, cycloalkyl, cycloalkenyl, and cycloalkynyl moieties.

As used herein, the term “alkyl” includes both straight, branched andcyclic alkyl groups. An analogous convention applies to other genericterms such as “alkenyl”, “alkynyl” and the like. Furthermore, as usedherein, the language “alkyl”, “alkenyl”, “alkynyl” and the likeencompasses both substituted and unsubstituted groups.

The term “alkyl” refers to groups usually having one to eight,preferably one to six carbon atoms. For example, “alkyl” may refer tomethyl, ethyl, n-propyl, isopropyl, cyclopropyl, butyl, isobutyl,sec-butyl, tert-butyl, cyclobutyl, pentyl, isopentyl tert-pentyl,cyclopentyl, hexyl, isohexyl, cyclohexyl, and the like. Suitablesubstituted alkyls include, but are not limited to, fluoromethyl,difluoromethyl, trifluoromethyl, 2-fluoroethyl, 3-fluoropropyl,hydroxymethyl, 2-hydroxyethyl, 3-hydroxypropyl, benzyl, substitutedbenzyl and the like.

The term “alkenyl” refers to groups usually having two to eight,preferably two to six carbon atoms. For example, “alkenyl” may refer toprop-2-enyl, but-2-enyl, but-3-enyl, 2-methylprop-2-enyl, hex-2-enyl,hex-5-enyl, 2,3-dimethylbut-2-enyl, and the like. The language“alkynyl,” which also refers to groups having two to eight, preferablytwo to six carbons, includes, but is not limited to, prop-2-ynyl,but-2-ynyl, but-3-ynyl, pent-2-ynyl, 3-methylpent-4-ynyl, hex-2-ynyl,hex-5-ynyl, and the like.

The term “cycloalkyl” as used herein refers specifically to groupshaving three to seven, preferably three to ten carbon atoms. Suitablecycloalkyls include, but are not limited to cyclopropyl, cyclobutyl,cyclopentyl, cyclohexyl, cycloheptyl and the like, which, as in the caseof other aliphatic or heteroaliphatic or heterocyclic moieties, mayoptionally be substituted.

The term “heteroaliphatic” as used herein refers to aliphatic moietieswhich contain one or more oxygen, sulfur, nitrogen, phosphorous orsilicon atoms, e.g., in place of carbon atoms. Heteroaliphatic moietiesmay be branched, unbranched or cyclic and include heterocycles such asmorpholino, pyrrolidinyl, etc.

The term “heterocycle” as used herein refers to cyclic heteroaliphaticand heteroaryl groups and preferably three to ten ring atoms total,includes, but is not limited to heteroaliphatic moieties such asoxetane, tetrahydrofuranyl, tetrahydropyranyl, aziridine, azetidine,pyrrolidine, piperidine, morpholine, piperazine and the like, andheteroaryl moieties as described below.

The terms “aryl” and “heteroaryl” as used herein refer to stable mono-or polycyclic, heterocyclic, polycyclic, and polyheterocyclicunsaturated moieties having 3-14 carbon atom which may be substituted orunsubstituted. Substituents include any of the previously mentionedsubstituents. Non-limiting examples of useful aryl ring groups includephenyl, halophenyl, alkoxyphenyl, dialkoxyphenyl, trialkoxyphenyl,alkylenedioxyphenyl, naphthyl, phenanthryl, anthryl, phenanthro and thelike. Examples of typical heteroaryl rings include 5-membered monocyclicring groups such as thienyl, pyrrolyl, imidazolyl, pyrazolyl, furyl,isothiazolyl, furazanyl, oxazolyl, isoxazolyl, thiazolyl, oxadiazolyland the like; 6-membered monocyclic groups such as pyridyl, pyrazinyl,pyrimidinyl, pyridazinyl, triazinyl and the like; and polycyclicheterocyclic ring groups such as benzo[b]thienyl, naphtho[2,3-b]thienyl,thianthrenyl, isobenzofuranyl, chromenyl, xanthenyl, phenoxathienyl,indolizinyl, isoindolyl, indolyl, indazolyl, purinyl, isoquinolyl,quinolyl, phthalazinyl, naphthyridinyl, quinoxalinyl, quinazolinyl,benzothiazole, benzimidazole, tetrahydroquinoline cinnolinyl,pteridinyl, carbazolyl, beta-carbolinyl, phenanthridinyl, acridinyl,perimidinyl, phenanthrolinyl, phenazinyl, isothiazolyl, phenothiazinyl,phenoxazinyl, and the like(see e.g. Katritzky, Handbook of HeterocyclicChemistry). The aryl or heteroaryl moieties may be substituted with oneto five members selected from the group consisting of hydroxy, C1-C8alkoxy, C1-C8 branched or straight-chain alkyl, acyloxy, carbamoyl,amino, N-acylamino, nitro, halo, trihalomethyl, cyano, and carboxyl.Aryl moieties thus include, e.g. phenyl; substituted phenyl bearing oneor more substituents selected from groups including: halo such as chloroor fluoro, hydroxy, C1-C6 alkyl, acyl, acyloxy, C1-C6 alkoxy (such asmethoxy or ethoxy, including among others dialkoxyphenyl moieties suchas 2,3-, 2,4-, 2,5-, 3,4- or 3,5-dimethoxy or diethoxy phenyl or such asmethylenedioxyphenyl, or 3-methoxy-5-ethoxyphenyl; or trisubstitutedphenyl, such as trialkoxy (e.g., 3,4,5-trimethoxy or ethoxyphenyl),3,5-dimethoxy-4-chloro-phenyl, etc.), amino, —SO₂NH₂, —SO₂NH(aliphatic),—SO₂N(aliphatic)₂, —O-aliphatic-COOH, and —O-aliphatic-NH₂ (which maycontain one or two N-aliphatic or N-acyl substituents).

A “halo” substituent may be fluoro, chloro, bromo or iodo.

With respect to nomenclature, note that asymetric moieties such as“—G—M—” are written in the direction or order in which they are intendedto be read into a given structure. Thus, “—G—M—” is distinct from“—M—G—”. For example, in “Ar—A—COOR”, where A is —G—M—, the structureAr—G—M—COOR, not Ar—M—G—COOR, is intended.

Synthesis

Those of ordinary skill in this art will appreciate that compounds ofthis invention may be produced using any of a variety of syntheticstrategies. We typically use a convergent synthetic scheme in which anintermediate comprising the desired “YXU” moiety, protected asappropriate, is condensed with a second intermediate comprising thedesired amino moiety HR¹⁴N(CR¹R²)_(m)B, again, protected as appropriate,to yield (following any necessary deprotection steps) the desiredcompound of Formula I. A variety of methods and materials for effectingthe relevant chemical transformations, product recovery, purificationand formulation are known in the art which may be adapted to use in thepractice of this invention. The detailed examples which followillustrate such syntheses and should provide helpful guidance to thepractitioner.

Assays for Comparative Functional Evaluation of Compounds

Compounds of this invention may be evaluated in a variety of assays todetermine their relative ability to bind to a receptor for apTyr-containing ligand, such as a protein containing one or more SH2 orPI domains, or to otherwise inhibit an intermolecular interactionmediated by such a domain. See e.g. U.S. Pat. No. 5,667,980 (Pawson;competitive binding assays), PCT/US97/02635 (Rickles et al; cell-basedassays) and PCT/US97/06746 (Lynch et al, FP assays). Compounds may alsobe evaluated for their selectivity of binding to one such receptor (orfamily of receptors) relative to another such receptor (or family ofreceptors). The compounds of this invention can be further evaluated byconventional methods for possible therapeutic applications, includingevaluations of toxicological and pharmacological activity. For example,the compounds may further be evaluated for activity in inhibitingcellular or other biological events mediated by a pathway involving themolecular interaction of interest using a suitable cell-based assay oran animal model. Cell-based assays and animal models suitable forevaluating inhibitory activity of a test compound with respect to a widevariety of cellular and other biological events are known in the art.New assays and models are regularly developed and reported in thescientific literature.

By way of non-limiting example, compounds which bind to an SH2 domaininvolved in the transduction of a signal leading to asthma or allergicepisodes may be evaluated in a mast cell or basophil degranulationassay. The inhibitory activity of a test compound identified as an SH2inhibitor by the method of this invention with respect to cellularrelease of specific mediators such as histamine, leukotrienes, hormonalmediators and/or cytokines, as well as its biological activity withrespect to the levels of phosphatidylinositol hydrolysis or tyrosinephosphorylation can be characterized with conventional in vitro assaysas an indication of biological activity. [See, e.g., Edward L. Barsumianet al, Eur. J. Immunol., 11:317-323 (1981); M. J. Forrest, Biochem.Pharmacol., 42:1221-1228 (1991) (measuring N-acetyl-betaglucosamin-adasefrom activated neutrophils); and Stephan et al., J. Biol. Chem.,267:5434-5441 (1992)].

For example, histamine release can be measured by a radioimmunoassayusing a kit available from AMAC Inc. (Westbrook, Me.). One can thusevaluate the biological activity of compounds of this invention andcompare them to one another and to known active compounds or clinicallyrelevant compounds which can be used as positive controls.

Generally speaking, in such assays IC₅₀ scores of 20 μM or less areconsidered of special interest, scores below 1 μM are considered ofparticular interest and scores below about 500 nM are of high interest.Inhibitors of this invention may also be tested in an ex vivo assay,e.g., for their ability to block antigen-stimulated contraction ofsensitized guinea pig tracheal strip tissue. Activity in this assay hasbeen shown to be useful in predicting the efficacy of potentialanti-asthma drugs.

Numerous animal models of asthma have been developed and can be used[for reviews, see Larson, “Experimental Models of Reversible AirwayObstruction”, in THE LUNG, Scientific Foundations, Crystal, West et al.(eds.), Raven Press, New York, pp. 953-965 (1991); Warner et al., Am.Rev. Respir. Dis., 141:253-257 (1990)]. Species used in animal models ofasthma include mice, rats, guinea pigs, rabbits, dogs, sheep andprimates. Other in vivo models available are described in Cross et al.,Lab Invest., 63:162-170 (1990); and Koh, et al., Science, 256:1210-1213(1992).

By way of further example, compounds which bind to an SH2 or otherdomain of interest involved in the transduction of a signal involved inthe initiation, maintenance or spread of cancerous growth may beevaluated in relevant conventional in vitro and in vivo assays. Seee.g., Ishii et al., J. Antibiot., XLII: 1877-1878 (1989); and U.S. Pat.No. 5,206,249 (issued Apr. 27, 1993).

Compounds which bind to a ZAP SH2 domain or which otherwise inhibitZAP-70-mediated signaling may be evaluated for immunosuppressiveactivity, e.g., in any of the well-known in vitro or in vivoimmunosuppression assays.

Compounds which bind to a Src SH2 domain or which otherwise inhibitSrc-mediated signaling may be evaluated for activity in a variety ofassays considered predictive of activity in treating or preventingosteoporosis. Such assays include the various pit assays and calvariaassays, among others. Illustrative assays are described below.

Murine Calvaria Assay

In osteoporosis, excessive bone resorption results in decreased bonedensity. In vivo and in vitro models of bone resorption are used tostudy the processes leading to osteoporosis. In vitro, fetal rat longbone and murine calvaria cultures are routinely used. Both modelsdisplay similar responses to parathyroid hormone (PTH), a physiologicalmodulator of bone resorption (Stern, P. H. and N. S. Krieger. Comparisonof fetal rat limb bones and neonatal mouse calvaria: effects ofparathyroid hormone and 1,25-dihydroxyvitamin D₃. Calcif. Tissue Int.35: 172-176,1983). The calvaria model of bone resorption can besuccessfully used to screen osteotropic compounds as has been previouslyshown (Green, J. R., K. Muller and K. Jaeggi. Preclinical pharmacologyof CGP 42'446, a new, potent, heterocyclic bisphosphonate compound. J.Bone Miner. Res. 9: 745-751, 1994.).

In one modification of the conventional calvaria model, calvaria are notlabeled with ⁴⁵Ca⁺⁺. Instead, calvarial calcium release into the mediais assessed using a microtiter calorimetric calcium assay. Thismodification can yield more consistent responses than the radioactivemethodology and provides results which are comparable to literaturevalues for ⁴⁵Ca⁺⁺ assays.

One calvaria culture model tests the ability of anti-resorptivecompounds to prevent resorption (prophylactic model). A second modeltests the ability of these compounds to terminate ongoing resorption(therapeutic model). Cytotoxicity may be assessed in both models using alactate dehydrogenase (LDH) assay. These in vitro models of boneresorption may be used for routine screening and evaluation of compoundsfor their ability to alter osteoclast-mediated bone resorption.

Media Preparation

Calcium free Dulbecco's Modified Eagle's Medium (DMEM) may be obtainedin a 5×solution (Specialty Media, D-012). A 1×solution is prepared usingultrafiltered water. A suitable media contains 15% heat inactivatedhorse serum (Sigma, H 1270). Calcium concentration is adjusted to 1.65to 1.83 mM using 0.2 M CaCl₂. Penicillin (100 U/ml) and streptomycin(0.1 mg/ml) are added to the final media preparation. Indomethacin isprepared to 0.5 mg/ml (1.397×10⁻⁷ M) in ethanol, and is added to analiquot of DMEM to produce a final concentration of 0.5 μM. Bovineparathyroid hormone (1-34) may be obtained from Bachem (PCAL 100). PTHis solubilized in 0.1% BSA and is then diluted in DMEM to produce afinal concentration of 10⁻⁶ M PTH. Ten-fold serial dilutions areperformed down to 10⁻¹¹ M.

Calvaria Dissection

Pregnant CD-1 mice may be obtained from Charles River and are subjectedto parturition. Neonatal mice (4-6 days) are cleansed with betadine andthen euthanized by decapitation. Adherent skin is cleared away from theskull, exposing the calvaria. The calvaria are dissected away from theskull using a 12B scalpel. Calvaria are immediately placed into a glasspetri dish containing room temperature Tyrode's Salt Solution (Sigma,T-2397). The calvaria are trimmed free of cartilage and bisected with ascalpel along the sagital suture. After dissection of all calvaria,calvaria are transferred into 24 well plates containing 0.5 μMindomethacin (Sigma, I-7378).

Culture Conditions

Calvaria are incubated in 1.5 ml DMEM in 24 well tissue culture platesat 37° C., 5% CO₂/air. Plates are rocked in the incubator using a Bellcorocker platform. Calvaria are pre-incubated in 0.5 μM indomethacin for24 hours. For each experiment, 6 to 8 random calvaria halves are usedfor each group. Both halves from a single mouse are never in the samegroup. Experiments are repeated at least three times.

Prophylactic Calvaria Experiment

After the 24 h pre-incubation period, calvaria are thoroughly washed inindomethacin-free DMEM. Calvaria are then transferred to new wellscontaining various PTH concentrations, and are cultured for anadditional 72 hours. Media samples (30 μl) are obtained every 24 hoursand assayed for calcium and LDH activity.

Therapeutic Calvaria Experiment

At the end of the 24 h pre-incubation period, the calvaria are washedfree of indomethacin using DMEM. Calvaria are then transferred to newwells containing DMEM or various concentrations of PTH. After 24 hourscalvaria are transferred into new wells with fresh media (PTH or DMEM)and cultured an additional 48 hours before addition of control vehicle.This may be accomplished by adding 3 μl of DMSO to new wells, andtransferring each calvaria along with its media into wells. Culturecontinues for a further 24 hours. Media samples are obtained after 72hours and 96 hours in culture with PTH and assayed for calcium.Additional samples are obtained after 48, 72, and 96 hours in culturewith PTH and assayed for LDH.

Calcium Assay

A commercially available diagnostic calcium assay (Sigma, No. 588-3),modified for use in a microtiter format, may be used to determinecirculating serum calcium concentrations. This colorimetric assay isdependent on the specific, high affinity complexation of calcium witharsenazo III dye under acidic conditions, which occurs with 1:1stoichiometry and absorbs at 600 nm (Bauer, P. J. Affinity andstoichiometry of calcium binding by Arsenazo III. Anal Biochem, 110:61,1981; Michaylova, V and P Ilkova. Photometric determination of microamounts of calcium with Arsenazo III. Anal Chim Acta, 53: 194, 1971).Magnesium has very low affinity for arsenazo III.

Briefly, 15 μl of media or rat sera (see below) is diluted 18-fold withultrafiltered water (nearly calcium-free). Fifty pi of this solution arepipetted into microtiter wells (Nunc, Maxisorp, flat-bottom, 0.4ml/well). Standards of 0, 0.5, 1, 2.5, 3.75, 5, 6.25, and 7.5 mg/dl (mg%) calcium, diluted 8-fold with ultrafiltered water from controlstandards (Sigma, 360-11), are used to construct standard curves. Onceall standards and samples are pipetted onto the plate, 150 μl ofdiagnostic reagent is added to initiate complexation. Optical densitymeasurements are obtained on a microtiter plate reader (MolecularDevices, ThermoMax) at 600 nm.

Lactate Dehydrogenase Assay

Phosphate buffer is prepared in distilled water (0.26 M K₂HPO₄.3H₂O,0.26 M KH₂PO₄; pH 7.4). A mix consisting of: 22 ml of phosphate buffer,6 ml distilled water and 2.0 ml of 0.01 M pyruvate is prepared. NADH isprepared to 0.4 mg/ml in phosphate buffer.

Ten μl of media samples obtained from incubated calvaria are added to 96well plates. Wells containing 10 μl of DMEM serve as blanks. To eachwell, 90 μl distilled water and 150 μl phosphate mix is added. 50 μlNADH is added using an eight channel pipette immediately before theplate is read on a microtiter plate reader at 340 nm. A kinetic assay isperformed for 10 minutes, with a read interval of 20 seconds.

Thyroid/Parathyroidectomized Rat Model of Bone Resorption

Parathyroid hormone (PTH) replacement in thyroparathyroidectomized(TPTX) rats is routinely used as an in vivo model of controlled boneresorption. Rats are the species of choice since the mechanisms of bonemodeling in the rat resemble those in humans. In addition, hormones andpharmacologic agents have similar effects on both rat and human bone(Frost, H. M. and W. S. S. Jee. On the rat model of human osteopeniasand osteoporoses. Bone and Mineral, 18: 227-236, 1992). Removal of thethyroid and parathyroid glands results in a rapid loss of parathyroidhormone (PTH) from the circulation. Since PTH inducesosteoclast-mediated bone resorption, this process is inhibited in TPTXanimals. In addition, PTH mediates calcium reabsorption from the kidneysand absorption from the small intestines. The lack of these activitieswork in concert to decrease serum calcium levels. In the absence of PTH,rats remain in a hypocalcemic state. Restriction of dietary calciumlimits intestinal calcium absorption and renal calcium filtration suchthat serum calcium levels are primarily influenced by bone resorption.Controlled PTH replacement therapy results in a controlled return ofserum calcium to baseline levels. When replacement occurs, concomitantlywith a low calcium diet, serum calcium increase is due to PTH-inducedosteoclast-mediated bone resorption. In this model, drugs which inhibitbone resorption prevent the PTH-mediated return of serum calcium tobaseline levels.

Female Wistar rats (226-250 gm, Charles River) are fasted overnight andanesthetized with 0.15 ml of 1.2% tribromoethanol (TBE). The ventralneck area is shaved and swabbed with betadine and isopropanol. A midlineincision is made in the neck through the skin and superficial musclelayer, as well as in the sternohyoid muscle. Blunt dissection isperformed to expose the thyroid gland. The thyroid gland is carefullyisolated from the trachea, thyrohyoid muscle, as well as adjacent nervesand blood vessels, using blunt dissection. The thyroid gland is excisedone lobe at a time. Cautery is performed for hemostasis. Care is takento avoid damaging the recurrent laryngeal nerve since damage to it isshown to affect serum calcium concentrations (Hirsch, P. F., G. F.Gauthier and P. L. Munson. Thyroid hypocalcemic principle and recurrentlaryngeal nerve injury as factors affecting the response toparathyroidectomy in rats. Endocrinology, 73: 244-252, 1963. et al.,1963). The incisions are closed using 3-0 vicryl. The wound is coatedwith triple antibiotic ointment (Fougera; 400 units/g bacitracin zinc, 5mg/g neomycin sulfate, 5000 units/g polymyxin B sulfate). FollowingTPTX, rats are pair fed a low calcium diet (Harlan Teklad TD 95065;<0.003% Ca⁺⁺, <0.04% PO₄) such that each rat receives the same quantityof food. Rats are fed at least 5 grams, but not more than 10 grams, offood. Rats consuming less than 3.0 grams of food receive the nutritionalsupplement Nutri-Cal p.o. (Evsco; <0.0033% calcium).

PTH Dose Response/Pump Implantation

Three days post TPTX, rats which are found to be hypocalcemic, based onday 2 serum calcium levels, are implanted with PTH-containing Alzetmini-osmotic pumps (ALZA, model 2001D) which pumps at a rate of 1 μl/h.The rats are anesthetized with ketamine (50 mg/kg, i.p.) andacepromazine (1.67 mg/kg, i.p.). The scapula region is shaved andprepared for surgery with betadine and isopropanol. A lateral incisionof approximately 2 cm in length is made between the scapulae. Usinghemostats, a subcutaneous pocket is created into which the Alzet pump isinserted. The wound is closed either with nylon suture or with staples.Triple antibiotic ointment is applied as described previously.

Bovine parathyroid hormone 1-34 (PTH) (Bachem California, PCAL 100) isprepared in vehicle (10⁻³ N HCl, 0.15 M NaCl, 20 mg/ml cysteine.HCl) atthe following concentrations: 0.156, 0.47, 1.56, 4.7, 15.6, and 156 μM.Alzet mini-osmotic pumps are filled with the PTH solution and maintainedin 37° C. saline for 4 hours prior to implantation.

Serum Samples

Rats are anesthetized by CO₂ from dry ice and daily blood samples areobtained via cardiac puncture using a 27 gauge needle. Baseline samplesare taken just prior to TPTX. Daily samples are obtained in the morning.Samples are allowed to clot on their side for several hours andsubsequently spun at 1000×g for 15 minutes to obtain serum. Serum isaliquoted and stored in the refrigerator until assayed for serumcalcium. Serum calcium is measured (see above) daily for at least 7 daysfollowing TPTX.

Uses of Compounds of this Invention

Compounds of this invention which bind to an SH2 domain of interest maybe used as biological reagents in assays as described herein forfunctional classification of a pTyr-binding domain (e.g. SH2 or PIdomain) of a particular protein, particularly a newly discoveredprotein. Families or classes of such proteins which bind topTyr-containing ligands may now be defined functionally, with respect toligand specificity. Moreover, compounds of this invention can be used toinhibit the occurrence of biological events resulting from molecularinteractions mediated by the protein of interest. Inhibiting suchinteractions can be useful in research aimed at better understanding thebiology of events mediated by the binding of pTyr-containing ligands totheir receptors.

Such compounds would be useful, for example, in the diagnosis,prevention or treatment of conditions or diseases resulting from acellular processes mediated by the binding of a pTyr-containing ligandwith a receptor therefor. For example, a patient can be treated toprevent the occurrence or progression of osteoporosis or to reverse itscourse by administering to the patient in need thereof an SH2inhibitorwhich selectively binds Src SH2 or otherwise interferes withSrc-mediated signaling.

There are many other conditions for which such signal transductioninhibitors may be useful therapeutically, including, e.g., breast cancerwhere the SH2 domain-containing proteins Src, PLCgamma and Grb7 havebeen implicated. Other relevant conditions include prostate cancer, inwhich case targeting Grb2, PLCgamma, and PI3K, all of which contain SH2domains, may be useful in treatment or prevention of the disease.Inhibition of the interaction of Grb2 or Abl SH2 domains with BCR-ablmay be useful to treat chronic myelogenous leukemia (CML) or acutemyelogenous leukemia (AML).

Still other relevant applications include the prevention of interferon-,growth factor-, or cytokine-mediated diseases (e.g. inflammatorydiseases) by targeting the interaction of STAT proteins with theirpTyr-containing ligands or otherwise inhibiting their signaltransduction pathways. Agents that block the SH2 domains of ZAP-70 orotherwise inhibit ZAP-70-mediated signaling would be candidates for thetreatment of immune-related disorders such as rejection of transplantedbone marrow, skin or other organs; rheumatoid arthritis; inflammatorybowel disease; and systemic lupus erythmatosis, and a variety ofautoimmune diseases.

By virtue of the capacity to inhibit protein-protein interactions or arelevant kinase or phosphatase activity required for cellular events ofpharmacologic importance, compounds of this invention which inhibitcellular signal transduction may be used in pharmaceutical compositionsand methods for treatment or prevention in a subject in need thereof.Such inhibitors can be used to treat or reduce the risk of the diseasesor their pathological effects mediated by such interactions.

For example, drugs that completely block one of the two ZAP SH2 domainsshould effectively prevent ZAP from associating with the activated TCRand thus block T cell activation. A ZAP antagonist or inhibitor wouldspecifically inhibit T cells and avoid the toxicity of the currentlyused immunosuppressive drugs, FK506 and cyclosporin, which target themore ubiquitously expressed protein, calcineurin. Since calcineurin isrequired for cellular activities in several tissues in addition to Tcells, cyclosporin and FK506 cause side effects in the kidney andcentral nervous system which limit their application largely to patientswith organ transplant rejection.

Therapeutic/Prophylactic Administration & Pharmaceutical Compositions

Compounds of this invention can exist in free form or, whereappropriate, in salt form. Pharmaceutically acceptable salts of manytypes of compounds and their preparation are well-known to those ofskill in the art. The pharmaceutically acceptable salts of compounds ofthis invention include the conventional non-toxic salts or thequaternary ammonium salts of such compounds which are formed, forexample, from inorganic or organic acids of bases.

The compounds of the invention may form hydrates or solvates. It isknown to those of skill in the art that charged compounds form hydratedspecies when lyophilized with water, or form solvated species whenconcentrated in a solution with an appropriate organic solvent.

This invention also relates to pharmaceutical compositions comprising atherapeutically (or prophylactically) effective amount of the compound,and a pharmaceutically acceptable carrier or excipient. Carriers includee.g. saline, buffered saline, dextrose, water, glycerol, ethanol, andcombinations thereof, and are discussed in greater detail below. Thecomposition, if desired, can also contain minor amounts of wetting oremulsifying agents, or pH buffering agents. The composition can be aliquid solution, suspension, emulsion, tablet, pill, capsule, sustainedrelease formulation, or powder. The composition can be formulated as asuppository, with traditional binders and carriers such astriglycerides. Oral formulation can include standard carriers such aspharmaceutical grades of mannitol, lactose, starch, magnesium stearate,sodium saccharine, cellulose, magnesium carbonate, etc. Formulation mayinvolve mixing, granulating and compressing or dissolving theingredients as appropriate to the desired preparation.

The pharmaceutical carrier employed may be, for example, either a solidor liquid.

Illustrative solid carrier include lactose, terra alba, sucrose, talc,gelatin, agar, pectin, acacia, magnesium stearate, stearic acid and thelike. A solid carrier can include one or more substances which may alsoact as flavoring agents, lubricants, solubilizers, suspending agents,fillers, glidants, compression aids, binders or tablet-disintegratingagents; it can also be an encapsulating material. In powders, thecarrier is a finely divided solid which is in admixture with the finelydivided active ingredient. In tablets, the active ingredient is mixedwith a carrier having the necessary compression properties in suitableproportions, and compacted in the shape and size desired. The powdersand tablets preferably contain up to 99% of the active ingredient.Suitable solid carriers include, for example, calcium phosphate,magnesium stearate, talc, sugars, lactose, dextrin, starch, gelatin,cellulose, methyl cellulose, sodium carboxymethyl cellulose,polyvinylpyrrolidine, low melting waxes and ion exchange resins.

Illustrative liquid carriers include syrup, peanut oil, olive oil,water, etc. Liquid carriers are used in preparing solutions,suspensions, emulsions, syrups, elixirs and pressurized compositions.The active ingredient can be dissolved or suspended in apharmaceutically acceptable liquid carrier such as water, an organicsolvent, a mixture of both or pharmaceutically acceptable oils or fats.The liquid carrier can contain other suitable pharmaceutical additivessuch as solubilizers, emulsifiers, buffers, preservatives, sweeteners,flavoring agents, suspending agents, thickening agents, colors,viscosity regulators, stabilizers or osmo-regulators. Suitable examplesof liquid carriers for oral and parenteral administration include water(partially containing additives as above, e.g. cellulose derivatives,preferably sodium carboxymethyl cellulose solution), alcohols (includingmonohydric alcohols and polyhydric alcohols, e.g. glycols) and theirderivatives, and oils (e.g. fractionated coconut oil and arachis oil).For parenteral administration, the carrier can also be an oily estersuch as ethyl create and isopropyl myristate. Sterile liquid carders areuseful in sterile liquid form compositions for parenteraladministration. The liquid carrier for pressurized compositions can behalogenated hydrocarbon or other pharmaceutically acceptable propellant.Liquid pharmaceutical compositions which are sterile solutions orsuspensions can be utilized by, for example, intramuscular,intraperitoneal or subcutaneous injection. Sterile solutions can also beadministered intravenously. The compound can also be administered orallyeither in liquid or solid composition form.

The carrier or excipient may include time delay material well known tothe art, such as glyceryl monostearate or glyceryl distearate along orwith a wax, ethylcellulose, hydroxypropylmethylcellulose,methylmethacrylate and the like. When formulated for oraladministration, 0.01% Tween 80 in PHOSAL PG-50 (phospholipid concentratewith 1,2-propylene glycol, A. Nattermann & Cie. GmbH) has beenrecognized as providing an acceptable oral formulation for othercompounds, and may be adapted to formulations for various compounds ofthis invention.

A wide variety of pharmaceutical forms can be employed. If a solidcarrier is used, the preparation can be tableted, placed in a hardgelatin capsule in powder or pellet form or in the form of a troche orlozenge. The amount of solid carrier will vary widely but preferablywill be from about 25 mg to about 1 g. If a liquid carrier is used, thepreparation will be in the form of a syrup, emulsion, soft gelatincapsule, sterile injectable solution or suspension in an ampule or vialor nonaqueous liquid suspension.

To obtain a stable water soluble dosage form, a pharmaceuticallyacceptable salt of the compound may be dissolved in an aqueous solutionof an organic or inorganic acid, such as a 0.3M solution of succinicacid or citric acid. Alternatively, acidic derivatives can be dissolvedin suitable basic solutions. If a soluble salt form is not available,the compound is dissolved in a suitable cosolvent or combinationsthereof. Examples of such suitable cosolvents include, but are notlimited to, alcohol, propylene glycol, polyethylene glycol 300,polysorbate 80, glycerin, polyoxyethylated fatty acids, fatty alcoholsor glycerin hydroxy fatty acids esters and the like in concentrationsranging from 0-60% of the total volume.

Various delivery systems are known and can be used to administer thecompound, or the various formulations thereof, including tablets,capsules, injectable solutions, encapsulation in liposomes,microparticles, microcapsules, etc. Methods of introduction include butare not limited to dermal, intradermal, intramuscular, intraperitoneal,intravenous, subcutaneous, intranasal, pulmonary, epidural, ocular and(as is usually preferred) oral routes. The compound may be administeredby any convenient or otherwise appropriate route, for example byinfusion or bolus injection, by absorption through epithelial ormucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa,etc.) and may be administered together with other biologically activeagents. Administration can be systemic or local. For treatment orprophylaxis of nasal, bronchial or pulmonary conditions, preferredroutes of administration are oral, nasal or via a bronchial aerosol ornebulizer.

In certain embodiments, it may be desirable to administer the compoundlocally to an area in need of treatment; this may be achieved by, forexample, and not by way of limitation, local infusion during surgery,topical application, by injection, by means of a catheter, by means of asuppository, or by means of a skin patch or implant, said implant beingof a porous, non-porous, or gelatinous material, including membranes,such as sialastic membranes, or fibers.

In a specific embodiment, the composition is formulated in accordancewith routine procedures as a pharmaceutical composition adapted forintravenous administration to human beings. Typically, compositions forintravenous administration are solutions in sterile isotonic aqueousbuffer. Where necessary, the composition may also include a solubilizingagent and a local anesthetic to ease pain at the side of the injection.Generally, the ingredients are supplied either separately or mixedtogether in unit dosage form, for example, as a lyophilized powder orwater free concentrate in a hermetically sealed container such as anampoule or sachette indicating the quantity of active agent. Where thecomposition is to be administered by infusion, it can be dispensed withan infusion bottle containing sterile pharmaceutical grade water orsaline. Where the composition is administered by injection, an ampouleof sterile water for injection or saline can be provided so that theingredients may be mixed prior to administration.

Administration to an individual of an effective amount of the compoundcan also be accomplished topically by administering the compound(s)directly to the affected area of the skin of the individual. For thispurpose, the compound is administered or applied in a compositionincluding a pharmacologically acceptable topical carrier, such as a gel,an ointment, a lotion, or a cream, which includes, without limitation,such carriers as water, glycerol, alcohol, propylene glycol, fattyalcohols, triglycerides, fatty acid esters, or mineral oils.

Other topical carriers include liquid petroleum, isopropyl palmitate,polyethylene glycol, ethanol (95%), polyoxyethylene monolaurate (5%) inwater, or sodium lauryl sulfate (5%) in water. Other materials such asanti-oxidants, humectants, viscosity stabilizers, and similar agents maybe added as necessary. Percutaneous penetration enhancers such as Azonemay also be included.

In addition, in certain instances, it is expected that the compound maybe disposed within devices placed upon, in, or under the skin. Suchdevices include patches, implants, and injections which release thecompound into the skin, by either passive or active release mechanisms.

Materials and methods for producing the various formulations are wellknown in the art and may be adapted for practicing the subjectinvention. See e.g. U.S. Pat. Nos. 5,182,293 and 4,837,311 (tablets,capsules and other oral formulations as well as intravenousformulations) and European Patent Application Publication Nos. 0 649 659(published Apr. 26, 1995; illustrative formulation for IVadministration) and 0 648 494 (published Apr. 19, 1995; illustrativeformulation for oral administration).

The effective dose of the compound will typically be in the range ofabout 0.01 to about 50 mg/kgs, preferably about 0.1 to about 10 mg/kg ofmammalian body weight, administered in single or multiple doses.Generally, the compound may be administered to patients in need of suchtreatment in a daily dose range of about 1 to about 2000 mg per patient.

The amount of compound which will be effective in the treatment orprevention of a particular disorder or condition will depend in part onthe nature and severity of the disorder or condition, which can bedetermined by standard clinical techniques. In addition, in vitro or invivo assays may optionally be employed to help identify optimal dosageranges. Effective doses may be extrapolated from dose-response curvesderived from in vitro or animal model test systems. The precise dosagelevel should be determined by the attending physician or other healthcare provider and will depend upon well known factors, including routeof administration, and the age, body weight, sex and general health ofthe individual; the nature, severity and clinical stage of the disease;the use (or not) of concomitant therapies.

The invention also provides a pharmaceutical pack or kit comprising oneor more containers filled with one or more of the ingredients of thepharmaceutical compositions of the invention. Optionally associated withsuch container(s) can be a notice in the form prescribed by agovernmental agency regulating the manufacture, use or sale ofpharmaceutical products, which notice reflects approval by the agency ofmanufacture, use or sale for human administration.

The representative examples which follow are intended to help illustratethe invention, and are not intended to, nor should they be construed to,limit the scope of the invention. Indeed, various modifications of theinvention and many further embodiments thereof, in addition to thoseshown and described herein, will become apparent to those skilled in theart from the full contents of this document, including the exampleswhich follow and the references to the scientific and patent literaturecited herein. It should further be appreciated that the contents ofthose cited references are incorporated herein by reference to helpillustrate the state of the art.

In addition, the full contents of U.S. patent applications U.S. Ser.Nos. 08/968,490 and 09/190,424(Weigele et al, “Novel Signal TransductionInhibitors, Compositions Containing Them & Uses Thereof” (filed Nov. 11,1998 and Nov. 11, 1999, respectively) and WO 99/24442, as well as U.S.Ser. Nos. 60/078,412 and 60/108,084 (Buchanan et al, “Novel SignalTransduction Inhibitors, Compositions Containing Them & Uses Thereof”,filed Mar. 18, 1998 and Nov. 12, 1998, respectively) and WO 99/47529 areincorporated by reference herein. Those documents provide additionalsynthetic and other guidance which may be of interest to thepractitioner of the subject invention.

The following examples contain important additional information,exemplification and guidance which can be adapted to the practice ofthis invention in its various embodiments and the equivalents thereof.

EXAMPLES Example 1{4-[(S)-2-Acetylamino-2-(3-carbamoyl-2-gyclohexylmethoxy-6,7,8,9-tetrahydro-5H-benzocyclohepten-(S)-5-ylcarbamoyl)-ethyl]-phenoxy}-aceticacid

(a) 2-Cyclohexylmethoxy-6,7,8,9-tetrahydro-5H-benzocycloheptene

To a mixture of 6,7,8,9-tetrahydro-5H-benzocyclohepten-2-ol (HelveticaChimica Acta 1947, 1883.) (9.9 g, 61.0 mmol) and Cs₂CO₃ (26.3 g, 73.2mmol) in DMF (100 ML) was added (bromomethyl)cyclohexane (10.2 mL, 73.2mmol). The mixture was heated to 60° C. for 18 h, dumped into water andextracted with ethyl acetate. The combined extracts were washed withwater, dried over magnesium sulfate and concentrated to a solid. Thesolid was recrystallized from ethanol (13.0 g, 83%). m.p. 62-63° C.

(b) 2-Cyclohexylmethoxy-6,7,8,9-tetrahydro-benzocyclohepten-5-one

A mixture of 2-cyclohexylmethoxy-6,7,8,9-tetrahydro-5H-benzocycloheptene(0.50 g, 1.94 mmol), potassium persulfate (1.57 g, 5.82 mmol),copper(II) sulfate pentahydrate (0.48 g, 1.94 mmol) and CH₃CN/H₂O (1:1,13 mL) were heated at reflux for 45 min. After cooling the mixture wasdiluted with water and extracted with EtOAc. The combined extracts werewashed with water, dried over magnesium sulfate, concentrated, andchomatographed over silica gel (20% EtOAc/hexane) to a white solid (0.46g, 87%). MS [M+H]⁺ 273, m.p. 86-88° C.

(c) 2-Cyclohexylmethoxy-6,7,8,9-tetrahydro-5H-benzocyclohepten-5-ol

To a suspension of2-cyclohexylmethoxy-6,7,8,9-tetrahydro-benzocyclohepten-5-one (14.5 g,53.23 mmol) in EtOH (150 mL) was added NaBH₄ (1.96 g, 53.23 mmol)portionwise over 10 min. The mixture was heated to 40° C. for 1 h,cooled to 0° C., then made acidic with the addition of 1 N HCl. Thewhite precipated solids were filtered, washed with water, and dried invacuuo (14.25 g, 97%). m.p. 102-103° C.

(d) 5-Azido-2-cyclohexylmethoxy-6,7,8,9-tetrahydro-5H-benzocycloheptene

To 2-cyclohexylmethoxy-6,7,8,9-tetrahydro-5H-benzocyclohepten-5-ol (14.2g, 52.0 mmol) in toluene (100 mL) at 0° C. was added DBU (9.4 mL, 62.9mmol) followed by diphenylphosphoryl azide (13.6 mL, 62.9 mmol). Themixture was allowed to stir for 18 h at rt, diluted with EtOAc, thenwashed successively with 1 N HCl, and water. The organic layer was driedover MgSO₄, filtered, concentrated, and chomatographed over silica gel(5% EtOAc/hexane) to yield a 7:3 mixture of5-azido-2-cyclohexylmethoxy-6,7,8,9-tetrahydro-5H-benzocycloheptene/3-cyclohexylmethoxy-6,7-dihydro-5H-benzocycloheptene(12.6 g). This mixture was used without further purification in the nextreaction.

(e)(2-Cyclohexylmethoxy-6,7,8,9-tetrahydro-5H-benzoclohepten-5-yl)-carbamicacid tert-butyl ester

To the mixture from the preceeding reaction dissolved in EtOAc (150 mL)was added 10% Pd/C (500 mg) followed by Boc₂O (5.61 g, 25 mmol). Themixture was hydrogenated at STP for 17 h, filtered though glass,concentrated, and chomatographed over silica gel to a white solid (7.93g, 41% from (c)). MS [M−H]⁻ 372, m.p. 125-126° C.

(f)(3-Bromo-2-cyclohexylmethoxy-6,7,8,9-tetrahydro-5H-benzocyclohepten-5-yl)-carbamicacid tert-butyl ester

To(2-cyclohexylmethoxy-6,7,8,9-tetrahydro-5H-benzocyclohepten-5-yl)-carbamicacid tert-butyl ester (7.93 g, 21.2 mmol) in CH₃CN (300 mL) was addedNBS (3.96 g, 22.3 mmol). The mixture was stirred for 1.5 h, then blowndry under a stream of N₂. The residue was resuspended in CCl₄, filtered,and concentrated to a solid. The solid was recrystallized from CH₃CN toyield a white solid (8.5 g, 88%). MS [M−H]⁻ 452, m.p. 142-144° C.

(g)9-tert-Butoxycarbonylamino-3-cyclohexylmethoxy-6,7,8,9-tetrahydro-5H-benzocycloheptene-2-carboxylicacid

To(3-bromo-2-cyclohexylmethoxy-6,7,8,9-tetrahydro-5H-benzocyclohepten-5-yl)-carbamicacid tert-butyl ester (8.0 g, 17.7 mmol) in THF (100 mL) at −78° C. wasadded nBuLi (21.2 mL, 53.1 mmol, 2.5 M hexane) dropwise. The mixture wasstirred for 10 min then a stream of CO₂ (g) was bubbled though themixture for 2 min. The mixture was diluted with ether and water, warmedto rt and the layers seperated. The aqueous layer was extracted withEt₂O and the combined extracts were washed with water, dried overmagnesium sulfate, and concentrated to a solid. The solid wasrecrystallized from Et₂O to yield a white solid (5.0 g, 68%). MS [M−H]⁻416, m.p. 159-160° C.

(h)(3-Carbamoyl-2-cyclohexylmethoxy-6,7,8,9-tetrahydro-5H-benzocyclohepten-5-yl)-carbamicacid tert-butyl ester

To9-tert-butoxycarbonylamino-3-cyclohexylmethoxy-6,7,8,9-tetrahydro-5H-benzocycloheptene-2-carboxylicacid (1.5 g, 3.60 mmol) in CH₂Cl₂/DMF (4:1, 25 mL) was added HOBT (0.53g, 4.0 mmol) and EDC (0.76 g, 4.0 mmol) and the mixture allowed to stirfor 30 min. To this was added NH₄OH (27% aqueous, 0.30 mL, 4.0 mmol) andstirring was continued for 3 h. The mixture was diluted with 1 M HCl andextracted with EtOAc. The combined extracts were washed with water,sat'd NaHCO₃, dried over magnesium sulfate,and concentrated to a solid.The solid was recrystallized from Et₂O to yield a white solid (5.0 g,68%). MS [M−H]⁻ 415, m.p. 193-194° C.

(i)9-Amino-3-cyclohexylmethoxy-6,7,8,9-tetrahydro-5H-benzocycloheptene-2-carboxylicacid amide

To(3-carbamoyl-2-cyclohexylmethoxy-6,7,8,9-tetrahydro-5H-benzocyclohepten-5-yl)-carbamicacid tert-butyl ester (0.60 g, 144 mmol) in CH₂Cl₂ (8 mL) was added TFA(2 mL). The mixture was stirred for 1 h, evaporated under a stream ofN₂, diluted with CH₂Cl₂ and washed with 1 N NaOH. The organic layer wasdried over magnesium sulfate and concentrated to a tan solid (0.45 g,99%). MS [M+H]⁺ 316, m.p. 170-172° C.

(j)(R,S)-9-[(S)-2-Acetylamino-3-(4-hydroxy-phenyl)-propionylamino]-3-cyclohexylmethoxy-6,7,8,9-tetrahydro-5H-benzocycloheptene-2-carboxylicacid amide

To N-acetyl-L-tyrosine (0.12 g, 0.55 mmol) in 5 mL of DME at 0° C. wasadded HOBT (0.083 g, 0.55 mmol), EDC (0.10 g, 0.55 mmol) and9-amino-3-cyclohexylmethoxy-6,7,8,9-tetrahydro-5H-benzocycloheptene-2carboxylicacid amide (0.16 g, 0.50 mmol). The resulting solution was allowed tostir for 3 h. The clear, yellow solution was diluted with 75 mL of ethylacetate and was washed with water, 1 N HCl, saturated NaHCO₃, saturatedNH₄Cl and brine. Drying over MgSO₄ and concentration yielded a whitefoamy solid (0.24 g, 92%). MS [M+H]⁺ 521.

(k){4-[(S)-2-Acetylamino-2-(3-carbamoyl-2-cyclohexylmethoxy-6,7,8,9-tetrahydro-5H-benzocyclohepten-(R,S)-5-ylcarbamoyl)-ethyl]-phenoxy}-aceticacid tert-butyl ester

To(R,S)-9-[(S)-2-acetylamino-3-(4-hydroxy-phenyl)-propionylamino]-3-cyclohexylmethoxy-6,7,8,9-tetrahydro-5H-benzocycloheptene-2-carboxylicacid amide (0.10 g, 0.19 mmol) in DMF (2 mL) was added potassiumcarbonate (8.0 mg, 0.58 mmol) and tert-butyl bromoacetate (85 μL, 0.58mmol). The suspension was stirred at room temperature under anatmosphere of N₂ for 48 hours, after which the solution was diluted with25 mL ethyl acetate and washed with water, 1N HCl, and brine. Dryingover MgSO₄ and concentration yielded the crude ester which was usedwithout purification immediately in the next reaction.

(l){4-[(S)-2-Acetylamino-2-(3-carbamoyl-2-cyclohexylmethoxy-6,7,8,9-tetrahydro-5H-benzocyclohepten-(Ror S)-5-ylcarbamoyl)-ethyl]-phenoxy}-acetic acid

To{4-[(S)-2-acetylamino-2-(3-carbamoyl-2-cyclohexylmethoxy-6,7,8,9-tetrahydro-5H-benzocyclohepten-(R,S)-5-ylcarbamoyl)-ethyl]-phenoxy}-aceticacid tert-butyl ester (crude from the preceeding reaction) in methylenechloride (2 mL) was added TFA (0.25 mL). The mixture was stirred for 20min., evaporated to dryness, and dissolved in DMSO (2 mL). Purificationby RP HPLC (CH₃CN/H₂O) and lyophylization yielded two isomers: Isomer 1(S,S): white solid (9.6 mg, 8.5%). MS [M−H]⁺ 580. Isomer 2 (S,R): whitesolid (34 mg, 7.7%). MS [M−H]⁺ 580.

Example 2 Phosphoric acidmono-{4-[(S)-2-acetylamino-2-(3-carbamoyl-2-cyclohexylmethoxy-6,7,8,9-tetrahydro-5H-benzocyclohepten-(R,S)-5-ylcarbamoyl)-ethyl]-phenyl}ester

(a) Phosphoric acid4-[(S)-2-acetylamino-2-(3-carbamoyl-2-cyclohexylmethoxy-6,7,8,9-tetrahydro-5H-benzocyclohepten-(R,S)-5-ylcarbamoyl)-ethyl-phenylester dibenzyl ester

To(S)-9-[(S)-2-acetylamino-3-(4-hydroxy-phenyl)-propionylamino]-3-cyclohexylmethoxy-6,7,8,9-tetrahydro-5H-benzocycloheptene-2-carboxylicacid amide (0.13 g, 0.24 mmol) in CH₃CN at −20° C. were added insuccession DIEA (0.10 mL, 0.57 mmol), CCl₄ (0.15 mL, 1.55 mmol), DMAP(3.7 mg, 0.030 mmol), and dibenzylphosphite (0.10 mL, 0.45 mmol). Themixture was stirred for 3 h, diluted with water, and extracted withEtOAc. The combined extracts were washed with water, 1 N HCl, sat'dNaHCO₃, dried over NaSO₄, concentrated, and chomatographed over silicagel (5% MeOH/CH₂Cl₂) to give a semi-solid (0.12 g, 65%). MS [M+H]⁺ 782.

(b) Phosphoric acidmono-{4-[(S)-2-acetylamino-2-(3-carbamoyl-2-cyclohexylmethoxy-6,7,8,9-tetrahydro-5H-benzocyclohepten-(R,S)-5-ylcarbamoyl)-ethyl]-phenyl}ester

To phosphoric acid4-[(S)-2-acetylamino-2-(3-carbamoyl-2-cyclohexylmethoxy-6,7,8,9-tetrahydro-5H-benzocyclohepten-(R,S)-5-ylcarbamoyl)-ethyl]-phenylester dibenzyl ester (0.12 g, 0.16 mmol) in EtOH (5 mL) was added 20%Pd/C (100 mg). The mixture was hydrogenated at STP for 2 h. The mixturewas filtered though glass, concentrated and purified by RP HPLC(CH₃CN/H₂O). Lyophylization yielded a white solid (70 mg, 97%). MS[M−H]⁺ 602.

Example 3({4-[(S)-2-Acetylamino-2-(3-carbamoyl-2-cyclohexylmethoxy-6,7,8,9-tetrahydro-5H-benzocyclohepten-(Ror S)-5-ylcarbamoyl)-ethyl]phenyl}-difluoro-methyl)-phosphonic acid

(a)({4-[(S)-2-Acetylamino-2-(3-carbamoyl-2-cyclohexylmethoxy-6,7,8,9-tetrahydro-5H-benzocyclohepten-(Ror S)-5-ylcarbamoyl)-ethyl]-phenyl}-difluoro-methyl)-phosphonic aciddiethyl ester

To(S)-2-acetylamino-3-{4-[(diethoxy-phosphoryl)-difluoro-methyl]-phenyl}-propionicacid (Tetrahedron Lett. 1993, 34, 4125) (0.14 g, 0.38 mmol) inCH₂Cl₂/DMF (4:1, 4 mL) at 0° C. was added HOBT (0.063 g, 0.46 mmol) andEDC (0.085 g, 0.44 mmol). To this was added9-amino-3-Cyclohexylmethoxy-6,7,8,9-tetrahydro-5H-benzocycloheptene-2-carboxylicacid amide (0.11 g, 0.38 mmol) and stirring was continued for 1 h. Themixture was then diluted with EtOAc (70 mL), washed with 1 N HCl, sat'dNaHCO₃, sat'd NH₄Cl, and brine. The organic layer was dried over MgSO₄,filtered, and concentrated to a foamy solid (0.19 g, 84%). Purificationby RP HPLC (CH₃CN/H₂O) and lyophylization yielded two isomers: Isomer 1:white solid (52 mg, 20%). MS [M−H]⁺ 668. Isomer 2: white solid (90 mg,36%). MS [M−H]⁺ 668.

(b)({4-[(S)-2-Acetylamino-2-(3-carbamoyl-2-cyclohexylmethoxy-6,7,8,9-tetrahydro-5H-benzocyclohepten-(S)-5-ylcarbamoyl)-ethyl]-phenyl}-difluoro-methyl)-phosphonicacid

To Isomer 1 (preceeding reaction) (53 mg, 0.079 mmol) in CH₃CN (3 mL)cooled to −20° C. was added TMSI (0.15 mL, 1.0 mmol). The mixture wasstirred for 20 min, quenched with sat'd NaHCO₃ (5 mL), and purified byRP HPLC (CH₃CN/H₂O) to a white solid (39.4 mg, 78%). MS [M−H]⁺ 636.

(c)({4-[(S)-2-Acetylamino-2-(3-carbamoyl-2-cyclohexylmethoxy-6,7,8,9-tetrahydro-5H-benzocyclohepten-(R)-5-ylcarbamoyl)-ethyl]-phenyl}-difluoro-methyl)-phosphonicacid

Was made as for Isomer 1 (example 3, (b)). (62.3 mg, 70%). MS [M−H]⁺636.

Example 4({4-[(S)-2-Acetylamino-2-(3-carbamoyl-2-cyclohexylmethoxy-6,7,8,9-tetrahydro-5H-benzocyclohepten-(Sand R)-5-ylcarbamoyl)-ethyl]-phenyl}-phosphono-methyl)-phosphonic acid

(a) p(CH₂PO₃Et₂)-L-Phe-OH

Fmoc-p(CH₂PO₃Et₂)-L-Phe-OH (5.0 g, 9.3 mmol) was dissolved in 170 mL ofTHF and 50 mL of diethyl amine and the mixture was vigorously stirred atrt for 3 h. Solvents were removed under reduced pressure and the solidwas resuspended in anhydrous ether, filtered, and dried on high vacuumto afford 2.8 g (94%) of p(CH₂PO₃Et₂)-L-Phe-OH as white solid which wasused without purification in the next step.

(b) N-Boe-p(CH₂PO₃Et₂)-L-Phe-OH

To a solution of p(CH₂PO₃Et₂)-L-Phe-OH (5.0 g, 16.7 mmol) in a 1:1mixture of DME/water (140 ml) at 0° C. was added NaHCO₃ (3.1 g, 36.8mmol) followed by Boc2O (4.0 g, 18.4 mmol). The mixture was stirred at0° C. for 30 min then warmed to rt and stirred for 1 h. About 50 ml ofDME was removed by evaporation then the remaining aqueous solution wasextracted with EtOAc (2×50 mL). The aqueous layer was brought to pH 4with 1 N HCl and extracted with EtOAc (3×100 mL). The combined extracts(second) were washed with water, dried over MgSO₄, filtered andconcentrated to a colorless oil (6.2 g, 90%). MS [M−H]⁻ 414.

(c) N-Boc-p(CH₂PO₃Et₂)-L-Phe-OMe

To a solution of N-Boc-p(CH₂PO₃Et₂)-L-Phe-OH (5.1 g, 12.2 mmol) in DMF(60 mL) was added Cs₂CO₃ (4.8 g, 14.7 mmol) followed by MeI (0.76 ml,12.2 mmol). The mixture was stirred for 1 h, diluted with water (600 ml)and extracted with EtOAc (3×100 mL). The combined extracts were washedwith water, 10% NaHSO₃, dried over MgSO₄, filtered and concentrated to asolid which was recrystallized from EtOAc/hexane to give a white solid(4.6 g, 88%). MS [M−H]⁻ 428. m.p. 104-105° C.

(d) N-Boc-p[CH(PO₃Et₂)₂]-L-Phe-OMe

To a suspension of N-Boc-p(CH₂PO₃Et₂)-L-Phe-OMe (7.0 g, 16.3 mmol) in185 mL of anhydrous DME, purged with N₂ and cooled to −42° C. (CH₃CN/dryice), was added dropwise lithium bis(trimethylsilyl)amide (1 M THF, 48.9mL, 48.9 mmol). The reaction mixture was stirred at −42° C. for 15 min.Diethylchlorophosphate (4.7 mL, 32.6 mmol) was added and the orangesolution was stirred at −42° C. for an additional 20 min before beingquenched with 1 N HCl (20 ml). The mixture was further diluted withwater and extracted with EtOAc (3×100 mL). The combined extracts werewashed with water, dried over MgSO₄, filtered, concentrated, andchomatographed over silica gel (3% MeOH/CH₂Cl₂) to give a colorless oil(6.0 g, 65%). MS [M−H]⁻ 564.

(e) N-Boc-p[CH(PO₃Et₂)₂]-L-Phe-OH

To a solution of N-Boc-p[CH(PO₃Et₂)₂]-L-Phe-OMe (0.49 g, 0.966 mmol) in5 mL of THF cooled to 0° C. was added dropwise a solution of lithiumhydroxide monohydrate (49.0 mg, 1.17 mmol) in 1.0 mL of water. Thereaction mixture was stirred at 0° C. for 1 h. THF was removed underreduced pressure to a yellow oil which was diluted with 10 mL of 1 NHCl. The aqueous phase was extracted with CH₂Cl₂ (8×15 mL), and theextracts were combined, dried over Na₂SO₄, and concentrated to afford0.45 g (95%) of N-Boc-p[CH(PO₃Et₂)₂]-L-Phe-OH as a crystalline whitesolid. MS [M−H]⁻ 550. m.p. 84-87° C.

(f)[{4-[(S)-2-tert-Butoxycarbonylamino-2-(3-carbamoyl-2-cyclohexylmethoxy-6,7,8,9-tetrahydro-5H-benzcyclohepten-(R,S)-5-ylcarbamoyl)-ethyl]-phenyl}-(diethoxy-phosphoryl)-methyl]-phosphonicacid diethyl ester

To N-Boc-p[CH(PO₃Et₂)₂]-L-Phe-OH (0.22 g, 0.41 mmol) in CH₂Cl₂/DMF (4:1,4 mL) at 0° C. was added HOBT (0.066 g, 02.9 mmol) and EDC (0.092 g,0.48 mmol). To this was added9-amino-3-cyclohexylmethoxy-6,7,8,9-tetrahydro-5H-benzocycloheptene-2-carboxylicacid amide (0.12 g, 0.38 mmol) and stirring was continued for 1 h. Themixture was then diluted with EtOAc (70 mL), washed with 1 N HCl, sat'dNaHCO₃, sat'd NH₄Cl, and brine. The organic layer was dried over MgSO₄,filtered, and concentrated to a foamy solid (0.29 g, 90%). This materialwas used without further purification in the next step.

(g)[{4-[(S)-2-Amino-2-(3-carbamoyl-2-cyclohexylmethoxy-6,7,8,9-tetrahydro-5H-benzocyclohepten-(R,S)-5-ylcarbamoyl)-ethyl]-phenyl}-(diethoxy-phosphoryl)-methyl]-phosphonicacid diethyl ester

To the crude material from the preceeding reaction (0.29 g, 0.34 mmol)in CH₂Cl₂ (10 mL) was added HBFA (2 mL). The mixture was stirred for 30min, evaporated to dryness and purified by RP HPLC (CH₃CN/H₂O): Isomer1: white solid (143 mg, 50%). MS [M−H]⁺ 750. Isomer 2: white solid (88mg, 31%). MS [M−H]⁺ 750.

(h)[{4-[(S)-2-Acetylamino-2-(3-carbamoyl-2-cyclohexylmethoxy-6,7,8,9-tetrahydro-5H-benzocyclohepten-(S)-5-ylcarbamoyl)-ethyl]-phenyl}-(diethoxy-phosphoryl)-methyl]-phosphonicacid diethyl ester

To Isomer 1 (from the preceeding reaction) (0.14 g, 0.19 mmol) in CH₂Cl₂(2 mL) at 0° C. was added TEA (0.30 mL, 2.15 mmol) followed by Ac₂O(0.075 mL, 0.80 mmol). The mixture was stirred for 30 min, diluted withCH₂Cl₂, and washed with 1 N HCl, sat'd NaHCO₃, and sat'd NH₄Cl. Theorganic layer was dried over MgSO₄, filtered and concentrated to aglassy solid (79.2 mg, 53%). This material (which was homogeneous byHPLC) was used without purification in the next reaction.

(i)({4-[(S)-2-Acetylamino-2-(3-carbamoyl-2-cyclohexylmethoxy-6,7,8,9-tetrahydro-5H-benzocyclohepten-(S)-5-ylcarbamoyl)-ethyl]-phenyl}-phosphono-methyl)-phosphonicacid

To[{4-[(S)-2-acetylamino-2-(3-carbamoyl-2-cyclohexylmethoxy-6,7,8,9-tetrahydro-5H-benzocyclohepten-(S)-5-ylcarbamoyl)-ethyl]-phenyl}-(diethoxy-phosphoryl)-methyl]-phosphonicacid diethyl ester (0.079 g, 0.10 mmol) in CH₃CN at 0° C. was addedTMSI. The mixture was stirred for 30 min, quenched with sat'd NaHCO₃ (5mL) and purified by RP HPLC (CH₃CN/H₂O) to a white solid (61.2 mg, 90%).MS [M−H]⁺ 680.

(j)({4-[(S)-2-Acetylamino-2-(3-carbamoyl-2-cyclohexylmethoxy-6,7,8,9-tetrahydro-5H-benzocyclohepten-(R)-5-ylcarbamoyl)-ethyl]-phenyl}-phosphono-methyl)-phosphonicacid

Was made as for Isomer 1 (example 4, (steps h and i)). (62.3 mg, 70%).MS [M−H]⁺ 680.

Example 54-[(S)-2-Acetylamino-2-(3-carbamoyl-2-cyclohexylmethoxy-6,7,8,9-tetrahydro-5H-benzocyclohepten-(S)-5-ylcarbamoyl)-ethyl]-2-formyl-benzoicacid

(a) 3-(1,3-Dithiolan-2-yl)-N-acetyl-L-tyrosine methyl ester

To L-3-formyl-N-acetyltyrosine methyl ester (U.S. Pat. No. 4,022,910)(0.13 g, 0.48 mmol) in methylene chloride (5 mL) at 0° C. was addedboron trifluoride diethyl etherate (0.12 mL, 0.96 mmol) followed byethanedithiol (0.044 mL, 0.53 mmol). The mixture was allowed to warm tort and stirred for 1 h. The solution was dumped into water and thelayers separated. The aqueous layer was extracted with methylenechloride and the combined extracts were washed with water, dried overmagnesium sulfate and concentrated to a solid. The solid wasrecrystallized from ethyl acetate/hexane (0.14 g, 82%). m.p. 97-99° C.

(b)3-(1,3-Dithiolan-2-yl)-4-(trifluoromethansulfonyloxy)-N-acetyl-L-phenylalaninemethyl ester

To 3-(1,3-dithiolan-2-yl)-N-acetyltyrosine methyl ester (0.50 g, 1.46mmol) and triethylamine (0.22 mL, 1.61 mmol) in methylene chloride (10mL) at 0° C. was added N-phenyltrifluoromethanesulfonimide (0.58 g, 1.61mmol). The mixture was allowed to stir for five days then washedsequentially with 1 N NaOH, 1 N HCl, and brine. The organic layer wasdried over magnesium sulfate and concentrated to a solid. The solid wasrecrystallized from ethyl acetate/hexane (0.61 g, 87%). m.p. 116-118° C.

(c) 3-(1,3-Dithiolan-2-yl)-4-(carboxymethyl)-N-acetyl-L-phenylalaninemethyl ester

To3-(1,3-dithiolan-2-y)-4(trifluoromethansulfonyloxy)-N-acetyl-L-phenylalaninemethyl ester (0.51 g, 1.08 mmol) in DMSO/MeOH (3:2, 5 mL) was addedtriethylamine (0.33 mL, 2.36 mmol) followed by palladium acetate (0.0073g, 0.033 mmol) and 1,3-bis(diphenylphosphino)propane (0.013 g, 0.034mmol). Carbon monoxide was bubbled though for 3 min and the mixture washeated to 80° C. for 24 hours. After cooling, the solution was dilutedwith water and extracted with ethyl acetate. The combined extracts werewashed with water, dried over magnesium sulfate and concentrated to asolid. The solid was recrystallized from ethyl acetate/hexane (0.30 g,72%). m.p. 113-119° C.

(d) 3-(1,3-Dithiolan-2-yl)-4-(carboxymethyl)-N-acetyl-L-phenylalanine

To 3-(1,3-dithiolan-2-yl)-4(carboxymethyl)-N-acetyl-L-phenylalaninemethyl ester (0.22 g, 0.57 mmol) in THF (10 mL) at 0° C. was addedlithium hydroxide monohydrate (0.025 g, 0.60 mmol, 1 mL water). Themixture was stirred for 45 min, diluted with water, made acidic with 1 NHCl, and extracted with ethyl acetate. The combined extracts were washedwith water, dried over magnesium sulfate and concentrated to a glassysolid which was homogeneous by RP HPLC (0.18 g, 86%). MS [M−H]⁻ 368.

(e)4[(S)-2-Acetylamino-2-(3-carbamoyl-2-cyclohexylmethoxy-6,7,8,9-tetrahydro-5H-benzocyclohepten-(R,S)-5-ylcarbamoyl)-ethyl]-2-[1,3]dithiolan-2-yl-benzoicacid methyl ester

To 3-(1,3-dithiolan-2-yl)-4carboxymethyl)-N-acetyl-L-phenylalanine (0.13g, 0.35 mmol) in CH₂Cl₂/DMF (5:1, 5 mL) at 0° C. was added HOBT (0.051g, 0.35 mmol) and EDC (0.072 g, 0.35 mmol). The mixture was stirred for10 min then(R,S)-9-amino-3-cyclohexylmethoxy-6,7,8,9-tetrahydro-5H-benzocycloheptene-2-carboxylicacid amide (0.10 g, 0.32 mmol) was added and stirring was continued for1 h. The solution was dumped into water and the layers separated. Theaqueous layer was extracted with methylene chloride and the combinedextracts were washed with water, 1N HCl, dried over magnesium sulfate,and concentrated to a glassy solid (0.20 g, 99%) which was homogeneousby RP HPLC. MS [M−H]⁻ 636.

(f)4-[(S)-2-Acetylamino-2-(3-carbamoyl-2-cyclohexylmethoxy-6,7,8,9-tetrahydro-5H-benzocyclohepten-(R,S)-5-ylcarbamoyl-ethyl]-2-formyl-benzoicacid methyl ester

To4-[(S)-2-acetylamino-2-(3-carbamoyl-2-cyclohexylmethoxy-6,7,8,9-tetrahydro-5H-benzocyclohepten-(R,S)-5-ylcarbamoyl)-ethyl]-2-[1,3]dithiolan-2-yl-benzoicacid methyl ester (0.20 g, 0.31 mmol) in CHCl₃/MeOH (1:1, 6 mL) wasadded mercury (II) perchlorate hydrate (0.38 g, 0.94 mmol). The mixturewas stirred for 5 min then filtered though Celite (CHCl₃ wash). Theorganic layer was washed with 1 N HCl, saturated NaHCO₃, dried overmagnesium sulfate, filtered, and concentrated to a glassy solid (0.18 g)which was used immediately in the next reaction.

(g)4-[(S)-2-Acetylamino-2-(3-carbamoyl-2-cyclohexylmethoxy-6,7,8,9-tetrahydro-5H-benzocyclohepten-(S)-5-ylcarbamoyl)-ethyl]-2-formyl-benzoicacid

To4-[(S)-2-acetylamino-2-(3carbamoyl-2-cyclohexylmethoxy-6,7,8,9-tetrahydro-5H-benzocyclohepten-(R,S)-5-ylcarbamoyl)-ethyl]-2-formyl-benzoicacid methyl ester (0.18 g, 0.32 mmol) in THF (5 mL) was added lithiumhydroxide monohydrate (0.018 g, 0.42 mmol, 1 mL water). The mixture wasstirred for 1 h, acidified with TFA, and evaporated. The residue wasdiluted with DMSO (2 mL) and purified by RP HPLC (CH₃CN/H₂O).Lyophilization left a white solid (0.040 mg, 21%). MS [M−H]⁻ 576.

Example 6{4-[(S)-2-Acetylamino-2-(3-carbamoyl-2-cyclohexylmethoxy-6,7,8,9-tetrahydro-5H-benzocyclohepten-(S)-5-ylcarbamoyl)-ethyl]-2-phosphono-phenyl}-phosphonicacid

(a) 2-tert-Butoxycarbonylamino-3-(3,4-dihydroxy-phenyl)-propionic acidmethyl ester

To (3,4-dihydroxyphenyl)-L-alanine methyl ester hydrochloride (5.4 g,25.4 mmol) and Di-tert-butyl dicarbonate (5.5 g, 25.4 mmol) in a mixtureof THF (20 mL) and water (20 mL) at rt was added sodium bicarbonate (3.2g, 38.1 mmol). The mixture was allowed to stirred for 16 h then washedwith water, and extracted with EtOAc. The organic layer was dried overmagnesium sulfate, and concentrated to a solid. The solid wasrecrystallized frome ethyl acetate/hexane (7 g, 88%). MS [M+H]⁺ 312.m.p. 132-135° C.

(b)3-(3,4-Bis-trifluoromethanesulfonyloxy-phenyl)-2-tert-butoxycarbonylamino-propionicacid methyl ester

To 2-tert-Butoxycarbonylamino-3-(3,4-dihydroxy-phenyl)-propionic acidmethyl ester (12 g, 38.6 mmol) and triethyl amine (13 mL, 88.7 mmol) inmethylene chloride (100 mL) at 0° C. was addedN-phenyl-bis(trifluoromethanesulfonimide) (31.6 g, 88.7 mmol). Themixture was allowed to stirred for two days then washed sequentiallywith 1 N NaOH, 1 N HCl, and brine. The organic layer was dried overmagnesium sulfate, concentrated to a solid. The solid was recrystallizedfrom dichloremethane/hexane. MS [M+Na]⁺ 598. m.p. 80-82° C.

(c)3-[3,4-Bis-(dietho-phosphoryl)-phenyl]-2-tert-butoxycarbonylamino-propionicacid methyl ester

To3-(3,4-bis-trifluoromethanesulfonyloxy-phenyl)-2-tert-butoxycarbonylamino-propionicacid methyl ester (2 g, 3.47 mmol), diethyl phosphite (1 mL, 7.65 mmol)and 4-methyl morpholine (0.93 mL, 8.3 mmol) in MeCN (10 ml) was addedPd(PPh₃)₄ (167 mg, 0.15 mmol). The mixture was allowed to stirred fortwo days at 95° C. then diluted with saturated NH₄Cl and extracted withEtOAc. The organic layer was dried over magnesium sulfate, concentrated,and chomatographed over silica gel (5% MeOH/EtOAc) to an oil (0.2 g, 37%yield). MS [M+H]⁺ 552 & [M+Na] 574.

(d)3-[3,4-Bis-(diethoxy-phosphoryl)-phenyl]-2-tert-butoxycarbonylamino-propionicacid

To a solution of3-[3,4-Bis-(diethoxy-phosphoryl)-phenyl]-2-tert-butoxycarbonylamino-propionicacid methyl ester (110 mg, 0.2 mmol) in 5 mL of THF cooled to 0° C. wasadded dropwise a solution of lithium hydroxide monohydrate (8.5 mg, 0.2mmol) in 1.0 mL of water. The reaction mixture was stirred at 0° C. for1 h. THF was removed under reduced pressure to a yellow oil which wasdiluted with 10 mL of 1 N HCl. The aqueous phase was extracted withCH₂Cl₂ (2×15 mL), and the extracts were combined, dried over Na₂SO₄, andconcentrated to afford an oil 107 mg (100%). MS [M−H]⁻ 537.

(f)[4-[(S)-2-Amino-2-(3-carbamoyl-2-cyclohexylmethoxy-6,7,8,9-tetrahydro-5H-benzocyclohepten-(S)-5-ylcarbamoyl)-ethyl]-2-(diethoxy-phosphoryl)-phenyl]-phosphonicacid diethyl ester

To3-(S)-[3,4-bis-(diethoxy-phosphoryl)-phenyl]-2-(2,2-dimethyl-propionylamino)-propionicacid (107 mg, 0.19 mmol) in a mixture of methylene chloride/DMF (5:1, 10mL) at 0° C. was added HOBt (27 mg, 0.2 mmol) and EDC (27 mg, 0.2 mmol).The mixture was stirred for 10 min then(R,S)-9-amino-3-cyclohexylmethoxy-6,7,8,9-tetrahydro-5H-benzocycloheptene-2-carboxylicacid amide (63 mg, 0.19 mmol) was added and stirring was continued for 1h. The solution was dumped into water and the layers separated. Theaqueous layer was extracted with methylene chloride and the combinedextracts were washed with water, dried over magnesium sulfate andconcentrated to a glassy solid. This crude material was dissolved inmethylene chloride (10 mL) and 95% aqueous TEFA (2 mL). The solution wasstirred for 1 h and then concentrated under a stream of N₂. The residuewas purified by preparative RP HPLC. Elution with 40:60 MeCN—H₂O (eachcontaining 0.1% TFA) provided the 60 mg of the title compound. MS [M+H]⁺736.

(g)[4-[(S)-2-Acetylamino-2-(3-carbamoyl-2-cyclohexylmethoxy-6,7,8,9-tetahydro-5H-benzocyclohepten-(S)-5-ylcarbamoyl)-ethyl]-2-(diethoxy-phosphoryl)-phenyl]-phosphonicacid diethyl ester

To[4-[(S)-2-amino-2-(3carbamoyl-2-cyclohexylmethoxy-6,7,8,9-tetrahydro-5H-benzocyclohepten-(S)-5-ylcarbamoyl)-ethyl]-2-(diethoxy-phosphoryl)-phenyl]-phosphonicacid diethyl ester (60 mg, 0.08 mmol) and triethyl amine (0.1 mL, 0.8mmol) in methylene chloride (10 mL) at 0° C. was added acetic anhydride(0.03 mL, 0.32 mmol). The mixture was allowed to stirred for 1 h thendiluted with water and extracted with EtOAc. The combined extracts weredried over magnesium sulfate and concentrated to a oil which washomogeneous by RP HPLC (65 mg, crude).

(h){4-[(S)-2-Acetylamino-2-(3-carbamoyl-2-cyclohexylmethoxy-6,7,8,9-tetrahydro-5H-benzocyclohepten-(S)-5-ylcarbamoyl)-ethyl]-2-phosphono-phenyl}-phosphonicacid

To a solution of[4-[(S)-2-acetylamino-2-(3-carbamoyl-2-cyclohexylmethoxy-6,7,8,9-tetrahydro-5H-benzocyclohepten-(S)-5-ylcarbamoyl)-ethyl]-2-(diethoxy-phosphoryl)-phenyl]-phosphonicacid diethyl ester (65 mg, 0.12 mmol) in MeCN (5 mL) at −11° C. wasadded TMSI (0.2 mL, 1.6 mmol). The mixture was stirred for 3 h at −11°C. and then quenched by saturated NaHCO₃ (1 mL). The resulting mixturewas purified by RP HPLC (CH₃CN/H₂O). Lyophilization left a white soild.MS [M−H]⁻ 664.

Example 7{4-[(S)-2-Amino-2-(3-carbamoyl-2-cyclohexylmethoxy-6,7,8,9-tetrahydro-5H-benzocyclohepten-(Ror S)-5-ylcarbamoyl)-ethyl]-2-phosphono-phenyl}-phosphonic acid

The title compounds were made from intermediates in the synthesis ofexample 6. MS [M−H]⁻ 622.

Example 8({4-[(S)-2-Acetylamino-2-(7-carbamoyl-8-cyclohexylmethoxy-2,3,4,5-tetrahydro-benzo[b]oxepin-(S)-5-ylcarbamoyl)-ethyl]-phenyl}-phosphono-methyl)-phosphonicacid

(a) 4-(4-Carbamoyl-3-hydroxy-phenoxy)-butyric acid ethyl ester

To 2,4-dihydroxybenzamide (16.5 g, 108.3 mmol) in DMF (100 mL) was addedCs₂CO₃ (39.0 g, 119.1 mmol) followed by ethyl 4-bromobutyrate (17.0 mL,119.1 mmol). The mixture was heated to 70° C. for 2 h, cooled, dilutedwith water and extracted with EtOAc. The combined extracts were washedwith water, dried over magnesium sulfate, concentrated, andchomatographed over silica gel (60% EtOAc/hexane) to a white solid (4.2g, 15%). MS [M+H]⁻ 267.

(b) 4-(4-Carbamoyl-3-cyclohexylmethoxy-phenoxy)-butyric acid ethyl ester

To 4-(4-carbamoyl-3-hydroxy-phenoxy)-butyric acid ethyl ester (3.0 g,11.22 mmol) in DMF (25 mL) was added Cs₂CO₃ (5.11 g, 15.71 mmol)followed by (bromomethyl)cyclohexane (1.72 mL, 12.34 mmol). The mixturewas heated to 60° C. for 6 h, then dumped onto ice/H₂O. The formedsolids were collected by filtration (H₂O wash)and dried in vacuuo (3.74g, 92%). MS [M+H]⁺ 364. m.p. 98-99° C.

(c) 444-Carbamoyl-3-cyclohexylmethoxy-phenoxy)-butyric acid

To 4-(4-carbamoyl-3-cyclohexylmethoxy-phenoxy)-butyric acid ethyl ester(3.74 g, 10.29 mmol) in THF (20 mL) was added LiOH·H₂O (0.61 g, 14.4mmol) and H₂O (6 mL). The mixture was stirred for 30 min. concentrated,diluted with water, and acidified with 1 N HCl. White solids werefiltered, washed with water, and dried in vacuuo (3.37 g, 95%). MS[M+H]⁺ 336.

(d)8-Cyclohexylmethoxy-5-oxo-2,3,4,5-tetrahydro-benzo[b]oxepine-7-carboxylicacid amide

4-(4-Carbamoyl-3-cyclohexylmethoxy-phenoxy)-butyric acid (3,4 g, 10.6mmol) was added to PPA at 80° C. The mixture was stirred for 1 h thendumped onto ice. The formed solids were filtered, washed with water,dried, and further purified on silica gel (5% MeOH/CH₂Cl₂) to give anoff-white solid (2.62 g, 82%). MS [M+H]⁺ 364. m.p. 185-186° C.

(e)8-Cyclohexylmethoxy-(R)-5-hydroxy-2,3,4,5-tetrahydro-benzo[b]oxepine-7-carboxylicacid amide

To8-cyclohexylmethoxy-5-oxo-2,3,4,5-tetrahydro-benzo[b]oxepine-7carboxylicacid amide (4.0 g, 13.3 mmol) suspended in CH₂Cl₂ (25 mL) at 42° C. wasadded (+)-DIP-Chloride (25 mL of CH₂Cl₂). The mixture was stirred at−12° C. for 18 h, concentrated, diluted with Et₂O (200 mL) and treatedwith diethanolamine (5.1 mL, 53.1 mmol). After stirring for 2.5 h, theformed solids were filtered and washed with Et₂O. The filtrate wasconcentrated, and the residue purified by silica gel columnchomatography (5% MeOH/CH₂Cl₂) to a white solid (3.2 g, 80%). m.p.164-165° C. Optical purity determined by Mosher Amide formation, 71%e.e.

(f)(S)-5-Azido-8-cyclohexylmethoxy-2,3,4,5-tetrahydro-benzo[b]oxepine-7-carboxylicacid amide

This compound was prepared as for Example 1, step d.

(g)(S)-5-Amino-8-cyclohexylmethoxy-2,3,4,5-tetrahydro-benzo[b]oxepine-7-carboxylicacid amide

To(S)-5-azido-8-cyclohexylmethoxy-2,3,4,5-tetrahydro-benzo[b]oxepine-7-carboxylicacid amide (2.5 g, 5.8 mmol) in EtOAc (20 mL) was added 10% Pd/C (0.25g). The mixture was hydrogenated at STP for 17 h, filtered though glass,and concentrated to a white solid (1.8 g, 98%). MS [M−H]⁻ 317, m.p.145-146° C. Optical purity determined by Mosher Amide formation, 55%e.e.

(h)({4-[(S)-2-Acetylamino-2-(7-carbamoyl-8-Cyclohexylmethoxy-2,3,4,5-tetrahydro-benzo[b]oxepin-(S)-5-ylcarbamoyl)-ethyl]-phenyl}-phosphono-methyl)-phosphonicacid

This compound was prepared as for Example 4. MS [M−H]⁻ 680.

Example 9({4-[2-(7-Carbamoyl-8-isobutoxy-2,3,4,5-tetrahydro-benzo[b]oxepin-(S)-5-ylcarbamoyl)-(S)-2-(2,2-dimethyl-propionylamino)-ethyl]-phenyl}-phosphono-methyl)-phosphonicacid

This compound was prepared as for Example 8. MS [M−H]⁺ 684.

Example 10({4-[2-(7-Carbamoyl-8-isobutoxy-2,3,4,5-tetrahydro-benzo[b]oxepin-(R)-5-ylcarbamoyl)-(S)-2-(2,2-dimethyl-propionylamino)-ethyl]-phenyl}-phosphono-methyl)-phosphonicacid

This compound was prepared as for Example 8. MS [M−H]⁺ 684.

Example 11({4-[(S)-2-Amino-2-(7-carbamoyl-8-cyclohexylmethoxy-2,3,4,5-tetrahydro-benzo[b]oxepin-(Ror S)-5-ylcarbamoyl)-ethyl]-phenyl}-phosphono-methyl)-phosphonic acid

The title compounds were made from intermediates in the synthesis ofexample 8. MS [M−H]⁻ 638

Example 12({4-[2-(7-Carbamoyl-8-cyclohexylmethoxy-2,3,4,5-tetrahydro-benzo[b]oxepin-(RorS)-5-ylcarbamoyl)-(S)-2-phenylacetylamino-ethyl]-phenyl}-phosphono-methyl)-phosphonicacid

These compound were prepared as for example 8. MS [M−H]⁺ 756.

Example 13{4-[(S)-2-Acetylamino-2-(7-carbamoyl-8-cyclohexylmethoxy-2,3,4,5-tetrahydro-benzo[b]oxepin-(Ror S)-5-ylcarbamoyl)-ethyl]-phenoxy}-acetic acid

(R,S)-5-Amino-8-cyclohexylmethoxy-2,3,4,5-tetrahydro-benzo[b]oxepine-7-carboxylicacid amide was converted to the title compounds as for example 1.

Example 14{4-[2-(7-Carbamoyl-8-isobutoxy-2,3,4,5-tetrahydro-benzo[b]oxepin-(S)-5-ylcarbamoyl)-(S)-2-(2,2-dimethyl-propionylamino)-ethyl]-2-phosphono-phenyl}-phosphonicacid

This compound was prepared using intermediates and procedures fromExample 6 and Example 8. MS [M−H]⁺ 670.

Example 15{4-[(S)-2-Amino-2-(7-carbamoyl-8-cyclohexylmethoxy-2,3,4,5-tetrahydro-benzo[b]oxepin-(S)-5-ylcarbamoyl)-ethyl]-2-phosphono-phenyl}-phosphonicacid

This compound was prepared using intermediates and procedures fromExample 6 and Example 8. MS [M−H]⁻ 624.

Example 16{4-[(S)-2-Acetylamino-2-(7-carbamoyl-8-cyclohexylmethoxy-2,3,4,5-tetrahydro-benzo[b]oxepin-(S)-5-ylcarbamoyl)-ethyl]-2-phosphono-phenyl}-phosphonicacid

This compound was prepared using intermediates and procedures fromExample 6 and Example 8. MS [M−H]⁻ 664.

Example 17{4-[2-(7-Carbamoyl-8-cyclohexylmethoxy-2,3,4,5-tetrahydro-benzo[b]oxepin-(S)-5-ylcarbamoyl)-2-phenylacetylamino-ethyl]-2-phosphono-phenyl}-phosphonicacid

This compound was prepared using intermediates and procedures fromExample 6 and Example 8. MS [M−H]⁻ 742.

Example 18{4-[(S)-2-Acetylamino-2-(7-carbamoyl-8-cyclohexylmethoxy-2,3,4,5-tetrahydro-benzo[b]oxepin-(S)-5-ylcarbamoyl)-ethyl]-phenyl}-phosphonicacid

This compound was prepared using an intermediate from Example 8 and fromBioorg Med. Chem. Lett. 1997, 7, 1909. MS [M−H]⁻ 586.

Example 19[(4-{(S)-2-Acetylamino-(S)-2-[1-(4-carbamoyl-7,8-dihydro-6H-5-oxa-9-thia-benzocyclohepten-2-yl)-ethylcarbamoyl]-ethyl}-phenyl)-difluoro-methyl]-phosphonicacid

(a) 2-(3-Hydroxy-propylsulfanyl)-phenol

2-Hydroxythiophenol (1.00 g, 8.70 mmol) was added to a mixture of DMF(10 mL) and Cs₂CO₃ (2.90 g, 8.90 mmol). To this was added3-bromopropanol (0.80 mL, 9.16 mmol) and the mixture was stirred for 20min. The mixture was added to into water and extracted with EtOAc. Thecombined extracts were washed with water, dried over magnesium sulfateand concentrated to a clear oil (2.36 g, 100%). MS [M−H]⁻ 183.

(b) 7,8-Dihydro-6H-5-oxa-9-thia-benzocycloheptene

To 2-(3-hydroxy-propylsulfanyl)-phenol (29.2 g, 158.5 mmol) in THF (450mL) was added triphenylphosphine (52.0 g, 200.0 mmol). The solution wascooled to −40° C. and diethyl azodicarboxylate (31.5 mL, 164.0 mmol) wasadded slowly. The solution was warmed to rt and stirred for 2.5 h. TheTHF was removed by evaporation and the residue was treated with 1 L ofEt₂O. The formed solids were filtered off, and the filtrate concentratedto an oil which was purified over silica gel (10% Et2O/hexane) to a pinkoil (16.2 g, 61%).

(c) 7,8-Dihydro-6H-5-oxa-9-thia-benzocycloheptene-4-carboxylic acid

To 7,8-dihydro-6H-5-oxa-9-thia-benzocycloheptene (16.2 g, 97 mmol) in250 mL of dry hexane was added tetramethylethylene diamine (16 mL, 106mmol). The solution was cooled to 0° C. and n-butyllitium (1.6 Msolution in hexane, 73 mL, 116.8 mmol) was slowly added with stirring. Atan-colored precipitate slowly started to form and some gas evolutionoccured. The suspension was stirred at rt for 18 h, after which CO₂ gaswas bubbled though it for 20 min. An exothermic reaction occured. Themixture was diluted with 300 mL ethyl acetate and 4 N HCl. After allsolids dissolved, the organic layer was washed with 1 N HCl. The acidwas extracted into the water layer using sat. NaHCO₃. The aqueous layerwas washed with ethyl acetate and treated with 10 N HCl to pH 1.Extraction with ethyl acetate (2×250 mL), drying over Na₂SO₄ andconcentration yielded the compound as a tan solid (16.5 g, 81%). MS[M−H]⁻ 209.

(d) 7,8-Dihydro-6H-5-oxa-9-thia-benzocycloheptene-4-carboxylic acidamide

To 7,8-dihydro-6H-5-oxa-9-thia-benzocycloheptene-4-carboxylic acid (16.5g, 78.4 mmol) in 100 mL DMF was added in succession solid HOBT (21.3 g,157.3 mmol), solid EDC hydrochloride (30.1 g, 157.0 mmol), and 25%aqueous ammonia (18 mL, 128.4 mmol). After stirring for 48 h, thereaction mixture was diluted with 200 mL ethyl acetate and washed withwater, 1N HCl, saturated NaHCO₃, saturated NH₄Cl, and brine. Drying overNa₂SO₄ and concentration yielded the amide as a tan solid (10.5 g, 64%).

(e) 5-Acetyl-3-(3-chloro-propylsulfanyl)-2-hydroxy-benzamide

Solid aluminum chloride (9.0 g, 67.7 mmol) was suspended in 20 mL of drydichloromethane at 0° C. The7,8-dihydro-6H-5-oxa-9-thia-benzocycloheptene-4-carboxylic acid amide(2.7 g, 12.7 mmol) was added as a solution in 20 mL dichloromethane. Thedeep green solution was stirred at 0° C. for 10 min, then neat acetylchloride (10 mL, 140.6 mmol) was added dropwise with stirring. Thesuspension was stirred at 0° C. for 20 min, then at rt for 30 h. Thereaction was quenched with 4 N HCl and extracted repeatedly with ethylacetate. Drying over Na2SO4 and concentration yeilded the crude product.Sgc (ethyl acetate) yielded the product as a tan solid (1.6 g, 44%). MS[M−H]⁻ 286.

(f) 2-Acetyl-7,8-dihydro-6H-5-oxa-9-thia-benzocycloheptene-4-carboxylicacid amide

The 5-acetyl-3-(3-chloro-propylsulfanyl)-2-hydroxy-benzamide (2.87 g, 10mmol) was dissolved in 8 mL dry DMF. Solid Cs₂CO₃ (4.93 g, 15.1 mmol)was added, followed by catalytic amounts of KI (0.1 g). The suspensionwas warmed to 70° C. under nitrogen and was stirred for 72 h. Aftercooling, it was diluted with ethyl acetate and enough 4 N HCl to makethe pH about 2. The aqueous layer was extracted with more ethyl acetate.The combined organic layers were washed with water and brine. Dryingover Na₂SO₄ and concentration yielded the product as a solid (1 g, 40%).

(g)2-(1-Hydro-ethyl)-7,8-dihydro-6H-5-oxa-9-thia-benzocycloheptene-4-carboxylicacid amide

To 2-acetyl-7,8-dihydro-6H-5-oxa-9-thia-benzocycloheptene-4-carboxylicacid amide (0.40 g, 1.59 mmol) suspended in EtOH (10 mL) was added NaBH₄(0.060 g, 1.59 mmol). The mixture was stirred for 5 min, made acidicwith 1 N HCl, and the EtOH removed in vacuuo. The aqueous was extractedwith EtOAc. The combined extracts were washed with water, dried overmagnesium sulfate and concentrated to a foam (0.35 g, 87%). MS M+H]⁺252.

(h)2-(1-Azido-ethyl)-7,8-dihydro-6H-5-oxa-9-thia-benzocycloheptene-4-carboxylicacid amide

This compound was prepared as for example 1 (d). (0.26 g, 66%).

(i)2-(1-Amino-ethyl-7,8-dihydro-6H-5-oxa-9-thia-benzocycloheptene-4-carboxylicacid amide

To2-(1-azido-ethyl)-7,8-dihydro-6H-5-oxa-9-thia-benzocycloheptene-4-carboxylicacid amide (0.20 g, 0.73 mmol) in THF (5 mL) was added water (0.10 mL)and triphenylphosphine (0.19 g, 0.73 mmol). The mixture was heated to50° C. for 20 h, evaporated, and chomatographed over silica gel (10%MeOH/CHCl₃) to give a colorless oil (0.10 g, 55%).

(j)[(4-{(S)-2-Acetylamino-(S)-2-[1-(4-carbamoyl-7,8-dihydro-6H-5-oxa-9-thia-benzocyclohepten-2-yl)-ethylcarbamoyl]-ethyl}-phenyl)-difluoro-methyl]-phosphonicacid

This compound was prepared as for example 3 (a-b). MS [M+H]⁺ 572.

Example 20{4-[(S)-2-Acetylamino-2-(3-carbamoyl-2-cyclohexylmethoxy-6,7,8,9-tetrahydro-5H-benzocyclohepten-(S)-5-ylcarbamoyl)-ethyl]-2-phosphono-phenoxy}-aceticacid and[4-[(S)-Acetylamino-2-(3-carbamoyl-2-cyclohexylmethoxy-6,7,8,9-tetahydro-5H-benzocyclohepten-(S)-5-ylcarbamoyl)-ethyl]-2-(ethoxy-hydroxy-phosphoryl)-phenoxy]-aceticacid

(a) Phosphoric acid diethyl ester 2-iodo-phenyl ester

To a mixture of 2-iodophenol (14.1 g, 64.1 mmol) and potassium carbonate(17.6 g, 128 mmol) in MeCN (100 mL) was added diethylchlorophosphate(11.1 mL, 76.7 mmol). The mixture was allowed to stir at rt for 5 h. Thesolvent was removed under reduced pressure, the residue diluted withwater and extracted with EtOAc. The organic layer was washed with brine,dried over anhydrous Na₂SO₄ and concentrated under reduced pressure. Theresidue was chromatographed over silica gel (elution with a stepwisegradient 25-40% EtOAc-hexanes) to give 21.7 g (95%) of a pale oil. R_(f)0.47 (1:1 EtOAc-hexanes). Electrospray Mass Spectrum (50/50acetonitrile/water) m/z 357 (M+H).

(b) (2-hydroxy-phenyl)-phosphonic acid diethyl ester Casteel, D. A.;Peri, S. P. Synthesis, 1991, 691.

To a cooled (−78° C.) solution of phosphoric acid diethyl ester2-iodo-phenyl ester (21.7 g, 61.0 mmol) in dry THF (500 mL) under N₂ wasadded a 2.5 M solution of BuLi (40 mL, 100 mmol). After 20 min, thereaction was treated with satd aq NH₄Cl (50 mL) and allowed to warm tort. The mixture was diluted with H₂O (50 mL) and extracted twice withEtOAc. The organic extracts were pooled, washed with brine, dried overanhydrous Na₂SO₄ and concentrated under reduced pressure. The residuewas chromatographed over silica gel (20% EtOAc-hexanes) to give 13.1 g(93%) of a pale oil. R_(f) 0.60 (1:1 EtOAc-hexanes). Electrospray MassSpectrum (50/50 acetonitrile/water) m/z 231 (M+H).

(c) (2-hydroxy-5-iodo-phenyl)-phosphonic acid diethyl ester

To a cooled (0° C.) mixture of (2-hydroxy-phenyl)-phosphonic aciddiethyl ester (13.1 g, 56.9 mmol) and sodium iodide (10.2 g, 68.3 mmol)in DMF (200 mL) was added chloramine-T trihydrate (19.2 g, 68.3 mmol)over 5 min. After 10 min, the reaction was allowed to warm to rt and themixture was allowed to stir at rt for 2 h. The mixture was diluted withH₂O (50 mL), acidified using 0.5 N HCl and extracted twice with EtOAc.The organic extracts were pooled and washed sequentially with satd.Na₂S₂O₃/brine (1/1) and then brine. The organic layer was dried overanhydrous Na₂SO₄ and concentrated under reduced pressure. The residuewas chromatographed over silica gel (elution with a stepwise gradient5-15% EtOAc-hexanes) to give 14.9 g (73%) of a colorless solid afterrecrystallization (EtOAc-hexanes). R_(f) 0.30 (20% EtOAc-hexanes).Electrospray Mass Spectrum (50/50 acetonitrile/water) m/z 357 (M+H).

(d) (2-benzyloxy-5-iodo-phenyl)-phosphonic acid diethyl ester

To a mixture of (2-hydroxy-5-iodo-phenyl)-phosphonic acid diethyl ester(14.7 g, 41.2 mmol) and cesium carbonate, (17.4 g, 53.5 mmol) in DMF(150 mL) was added benzyl bromide (6.40 mL, 54 mmol). The reaction wasallowed to stir for 2.5 days at rt at which point the reaction wasconcentrated under reduced pressure, the residue diluted in 0.5 N HCl(30 mL) and extracted twice with EtOAc. The pooled organic extracts werewashed with brine, dried over anhydrous Na₂SO₄ and concentrated underreduced pressure to give 17.4 g (91%) of a colorless solid afterrecrystallization (EtOAc-hexanes). R_(f) 0.36 (1:1 EtOAc-hexanes).Electrospray Mass Spectrum (50/50 acetonitrile/water) m/z 447 (M+H).

(e)3-[4-benzyloxy-3-(diethoxy-phosphoryl)-phenyl]-(S)-2-tert-butoxycarbonylamino-propionicacid methyl ester

To a mixture of Zn dust (403 mg, 6.17 mmol) in dry DMA (1 mL) and dryTHF (1 mL) was added dibromoethane (55 microliters, 0.64 mmol) andchlorotrimethylsilane (80 microliters, 0.63 mmol). The mixture wassonicated for 15 min under N₂. To this mixture was added a solution ofBoc-L-iodoalanine-methyl ester (1.45 g, 4.41 mmol) in dry DMA (1 mL) anddry THF (1 mL). The mixture was sonicated for 30 min and then heated(65° C.) for 45 min under N₂. To the heated mixture was added a solution(2-benzyloxy-5-iodo-phenyl)-phosphonic acid diethyl ester (1.37 g, 2.94mmol), bis(benzonitrile)dichloropalladium (II) (67 mg, 0.17 mmol) andtri-ortho-tolylphosphine (148 mg, 0.486 mmol) in dry DMA (1 mL) and THF(1 mL) over 5 min. The mixture was allowed to stir for 3 h at 65° C.under N₂ at which point the reaction was allowed to cool to rt, dilutedwith 0.5 N HCl and extracted twice with Et₂O. The organic extracts werepooled, washed with brine, filtered, dried over anhydrous MgSO₄ andconcentrated under reduced pressure. An initial chromatographicpurification over silica gel (elution with a stepwise gradient 15-40%acetone-hexanes) was followed by a second chromatographic purificationover silica gel (70% EtOAc-hexanes) to give 958 mg (63%) of a pale oil.R_(f) 0.33 (80% EtOAc-hexanes). Electrospray Mass Spectrum (50/50acetonitrile/water) m/z 522.5 (M+H).

(f)(S)-2-tert-butoxycarbonylamino-3-[4-benzyloxy-3-(diethoxy-phosphoryl)-phenyl]-propionicacid

To a cooled (0° C.) solution(S)-2-tert-butoxycarbonylamino-3-[3-(diethoxy-phosphoryl)-4-hydroxy-phenyl]-propionicacid methyl ester (423 mg, 0.98 mmol) in MeOH (4 mL) was added lithiumhydroxide monohydrate (84 mg, 2.0 mmol) in H₂O (4 mL). After 1 h, thereaction was allowed to warm to rt. After 2.5 h, the solution wascarefully acidified to pH 2 using 0.5 N HCl and extracted twice withEtOAc. The organic extracts were pooled, washed with brine, dried overanhydrous Na₂SO₄ and concentrated to give 379 mg (91%) of a yellow foam.The crude material was carried on without further purification.Electrospray Mass Spectrum (50/50 acetonitrile/water) m/z 416 (M−H).

(g)(S)-9-amino-3-cyclohexylmethoxy-6,7,8,9-tetrahydro-5H-benzocycloheptene-2-carboxylicacid amide

To a cooled (0° C.) solution of(S)-9-tert-butoxycarbonylamino-3-cyclohexylmethoxy-6,7,8,9-tetrahydro-5H-benzocycloheptene-2-carboxylicacid amide (198 mg, 0.475 mmol) in DCM (1 mL) was added TFA (1 mL).After 10 min, the reaction was allowed to warm to rt and after 1 h, thereaction was evaporated to dryness. The material was used withoutfurther purification for the next step.

(h){2-Benzyloxy-5-[(S)-2-tert-butoxycarbonylamino-2-(3carbamoyl-2-cyclohexylmethoxy-6,7,8,9-tetrahydro-5H-benzocyclohepten-(S)-5-ylcarbamoyl)-ethyl]-phenyl}-phosphonicacid diethyl ester

A solution of(S)-2-tert-butoxycarbonylamino-3-[4-benzyloxy-3-(diethoxy-phosphoryl)-phenyl]-propionicacid (355 mg, 0.700 mmol), 1-hydroxy-7-azabenzotriazole (123 mg, 0.904mmol) and(S)-9-amino-3-cyclohexylmethoxy-6,7,8,9-tetrahydro-5H-benzocycloheptene-2aboxylicacid amide (ca. 0.475 mmol) in DMF (5 mL) was neutralized by dropwiseaddition of 4-methylmorpholine. The solution was cooled (−20° C.) andEDC (173 mg, 903 mmol) was added. The reaction was allowed to slowlywarm to rt under N₂. After 16 h, the reaction was diluted with 0.5 N HCland extract ed twice with EtOAc. The pooled organic extracts were washedsequentially with brine, satd. NaHCO₃ and brine. The organic layer wasdried over Na₂SO₄ and concentrated. The residue was chromatographed oversilica gel (elution with 2% MeOH/CHCl₃) to give 342 mg (89%) of acolorless solid. R_(f) 0.59 (EtOAc). Electrospray Mass Spectrum (50/50acetonitrile/water) m/z 806 (M+H).

(i){5-[(S)-2-Acetylamino-2-(3-carbamoyl-2-cyclohexylmethoxy-6,7,8,9-tetrahydro-5H-benzocyclohepten-(S)-5-ylcarbamoyl)-ethyl]-2-benzyloxy-phenyl}-phosphonicacid diethyl ester

To a cooled (0° C.) solution of{2-benzyloxy-5-[(S)-2-tert-butoxycabonylamino-2-(3-carbamoyl-2-cyclohexylmethoxy-6,7,8,9-tetrahydro-5H-benzocyclohepten-(S)-5-ylcarbamoyl)ethyl]-phenyl}-phosphonicacid diethyl ester (342 mg, 0.425 mmol) in DCM (1 mL) was added TFA (1mL). After 10 min, the reaction was allowed to warm to rt and after 1 h,the reaction was evaporated to dryness. The crude material was dissolvedin DCM (3 mL) and 4-methylmorpholine (70 μL, 0.638 mmol) and aceticanhydride (60 μL, 0.638 mmol) were added. The reaction was allowed tostir at it for 17 h. The reaction was diluted with 0.5N HCl andextracted twice with EtOAc. The combined extracts were washed withbrine, dried over Na₂SO₄ and concentrated to a colorless oil. Thematerial was used without further purification for the next step.

(j)[4-[(S)-2-Acetylamino-2-(3-carbamoyl-2-cyclohexylmethoxy-6,7,8,9-tetrahydro-5H-benzocyclohepten-(S)-5-ylcarbamoyl)-ethyl]-2-(diethoxy-phosphoryl)-phenoxy]-aceticacid tert-butyl ester

To a solution of{5-[(S)-2-acetylamino-2-(3-carbamoyl-2-cyclohexylmethoxy-6,7,8,9-tetrahydro-5H-benzocycloheptene-(S)-5-ylcarbamoyl)-ethyl]-2-benzyloxy-phenyl}-phosphonicacid diethyl ester (ca. 0.425 mmol) in MeOH (10 mL) was added 10% Pd/C(4 mg). The heterogeneous mixture was degassed under reduced pressureand allowed to stir at rt for 18 h under H₂. The catalyst was removed byfiltration and the filtrate concentrated. The residue was dissolved inMeCN (5 mL) and cesium carbonate (0.64 mmoles) andtert-butylbromoacetate (0.64 mmoles) was added. The mixture was allowedto stir at rt for 17 h. The reaction was diluted with water andextracted twice with EtOAc. The combined extracts were washed withbrine, dried over Na₂SO₄ and concentrated. The residue waschromatographed over silica gel (elution with 2% MeOH/CHCl₃) to give 278mg (85%) of a colorless solid. R_(f) 0.21 (5% MeOH/CHCl₃). ElectrosprayMass Spectrum (50/50 acetonitrile/water) m/z 770 (M−H).

(k){4-[(S)-2-Acetylamino-2-(3-carbamoyl-2-cyclohexylmethoxy-6,7,8,9-tetrahydro-5H-benzocyclohepten-(S)-5-ylcarbamoyl)-ethyl]-2-phosphono-phenoxy}-aceticacid and[4-[(S)-Acetylamino-2-(3-carbamoyl-2-cyclohexylmethoxy-6,7,8,9-tetrahydro-5H-benzocyclohepten-(S)-5-ylcarbamoyl)-ethyl]-2-(ethoxy-hydroxy-phosphoryl)-phenoxy]-aceticacid

To a cooled (0° C.) solution of[4-[(S)-2-acetylamino-2-(3-carbamoyl-2-cyclohexylmethoxy-6,7,8,9-tetrahydro-5H-benzocyclohepten-(S)-5-ylcarbamoyl)-ethyl]-2-(diethoxy-phosphoryl)-phenoxy]-aceticacid tert-butyl ester (200 mg, 0.259 mmol) in DCM (1 mL) was added TFA(1 mL). After 10 min, the reaction was allowed to warm to rt and after 1h, the reaction was evaporated to dryness. The residue was dissolved inMeCN (3 mL) and cooled (0° C.). To this solution was added dropwiseiodotrimethylsilane (0.80 mL, 5.9 mmol). After 5 h at 0° C., thereaction was quenched with 20% NaHSO₃. A solution of 10% NaOH was addeduntil the mixture appeared colorless. The mixture was filtered andpurified by RP-HPLC (CH₃CN/H₂O) to give{4-[(S)-2-acetylamino-2-(3-carbamoyl-2-cyclohexylmethoxy-6,7,8,9-tetrahydro-5H-benzocyclohepten-(S)-5-ylcarbamoyl)-ethyl]-2-phosphono-phenoxy}-aceticacid (45 mg) MS 658 (M−H) and[4-[(S)-acetylamino-2-(3-carbamoyl-2-cyclohexylmethoxy-6,7,8,9-tetrahydro-5H-benzocyclohepten-(S)-5-ylcarbamoyl)-ethyl]-2-(ethoxy-hydroxy-phosphoryl)-phenoxy]-aceticacid (25 mg) MS 686 (M−H).

Example 21{4-[(S)-2-Acetylamino-2-(3-carbamoyl-2-cyclohexylmethoxy-6,7,8,9-tetrahydro-5H-benzocyclohepten-(S)-5-ylcarbamoyl)-ethyl]-2-carboxymethoxy-phenoxy}-aceticacid

(a)[1-(3-Carbamoyl-2-cyclohexylmethoxy-6,7,8,9-tetrahydro-5H-benzocyclohepten-(S)-5-ylcarbamoyl)-(S)-2-(3,4-dihydroxy-phenyl)-ethyl]-carbamicacid tert-butyl ester

(S)-2-tert-Butoxycarbonylamino-3-(3,4-dihydroxy-phenyl)-propionic aciddicyclohexylamine (125 mg, 0.300 mmol) was coupled to(S)-9-amino-3-cyclohexylmethoxy-6,7,8,9-tetrahydro-5H-benzocycloheptene-2-carboxylicacid amide as described for Example X (h). The product was obtained as acolorless foam (60 mg, 33%). R_(f) 0.32 (5% MeOH/CHCl₃). ElectrosprayMass Spectrum (50/50 acetonitrile/water) m/z 594 (M−H).

(b){4-[(S)-2-tert-Butoxycarbonylamino-2-(3-carbamoyl-2-cyclohexylmethoxy-6,7,8,9-tetrahydro-5H-benzocyclohepten-(S)-5-ylcarbamoyl)-ethyl]-2-tert-butoxycarbonylmethoxy-phenoxy}-aceticacid tert-butyl ester

To a solution of[1-(3-carbamoyl-2-cyclohexylmethoxy-6,7,8,9-tetrahydro-5H-benzocyclohepten-(S)-5-ylcarbamoyl)-(S)-2-(3,4-dihydroxy-phenyl)-ethyl]-carbamicacid tert-butyl ester (58 mg, 0.097 mmol) in DMF (1.5 mL) was addedtert-butylbromoacetate (50 μL, 0.34 mmol) and cesium carbonate (150 mg0.46 mmol). The mixture was allowed to stir at rt for 22 h. A secondaddition of tert-butylbromoacetate (25 μL, 0.17 mmol) and cesiumcarbonate (75 mg 0.23 mmol) was necessary to drive the reaction tocompletion after an additional 40 h. The mixture was diluted with EtOAcand 0.5 N HCl. The aqueous portion was extracted with fresh EtOAc andthe combined organic layers were washed with brine. The solution wasdried over Na₂SO₄, concentrated and chromatographed over silica gel(elution with 3% MeOH/CHCl₃) to give the product as a colorless solidafter trituration with EtOAc and hexanes (67 mg, 84%). R_(f) 0.11 (3%MeOH/CHCl₃). Electrospray Mass Spectrum (50/50 acetonitrile/water) m/z824 (M+H).

(c){4-[(S)-2-Acetylamino-2-(3-carbamoyl-2-cyclohexylmethoxy-6,7,8,9-tetrahydro-5H-benzocyclohepten-(S)-5-ylcarbamoyl)-ethyl]-2-carboxymethoxy-phenoxy}-aceticacid

To a cooled (0° C.) solution of{4-[(S)-2-tert-butoxycarbonylamino-2-(3-carbamoyl-2-cyclohexylmethoxy-6,7,8,9-tetrahydro-5H-benzocyclohepten-(S)-5-ylcarbamoyl)-ethyl]-2-tert-butoxycarbonylmethoxy-phenoxy}-aceticacid tert-butyl ester (66 mg, 0.050 mmol) in DCM (1 mL) was added TFA (1mL). After 0.5 h, the reaction was allowed to warm to rt and after 3 h,the reaction was evaporated to dryness. The crude material was dissolvedin DCM (1 mL) and the solution neutralized with 4-methylmorpholine.Acetic anhydride (10 μL, 0.11 mmol) was added and the reaction allowedto stir at rt for 18 h. The reaction was concentrated, diluted withMeCN/H₂O and purified by RP-HPLC (MeCN/H₂O) to give the product as acolorless solid after lyophilization (22 mg, 44%). Electrospray MassSpectrum (50/50 acetonitrile/water) m/z 654 (M+H).

Example 22{4-[(S)-2-Acetylamino-2-(3-carbamoyl-2-cyclohexylmethoxy-6,7,8,9-tetrahydro-5H-benzocyclohepten-(S)-5-ylcarbamoyl)-ethyl]-2-carboxymethyl-phenoxy}-aceticacid

(a) (S)-2-tert-Butoxycarbonylamino-3-(4-hydroxy-3-iodo-phenyl)propionicacid

To a solution of L-4-iodotyrosine (2.00 g, 6.51 mmol) in dioxane (50 mL)and H₂O (40 mL) was added 4-methylmorpholine (0.70 mL, 6.4 mmol) anddi-tert-butyldicarbonate (2.13 g, 9.77 mmol). After 18 h at rt,di-tert-butyldicarbonate (2.13 g, 9.77 mmol) was added to drive thereaction to completion. The reaction was allowed to stir for another 24h. The solution was acidified with 0.5 N HCl and extracted twice withEtOAc. The combined extracts were washed with brine, dried over Na₂SO₄and concentrated give the product as a foamy solid. The material wasused without further purification for the next step.

(b) (S)-2-tert-Butoxycarbonylamino-3-(4-hydroxy-3-iodo-phenyl)-propionicacid benzyl ester

To a solution of(S)-2-tert-butoxycarbonylamino-3-(4-hydroxy-3-iodo-phenyl)-propionicacid (3.27 g, 8.03 mmol) in DCM (20 mL) was added DMAP (1.03 g, 8.43mmol) and benzyl alcohol (2.4 mL, 23 mmol). The solution was cooled(−20° C.) and EDC (1.62 g, 8.45 mmol) was added. The reaction wasallowed to slowly warm to rt. After 17 h, the reaction was diluted with0.5 N HCl and extracted twice with EtOAc. The combined extracts werewashed with brine, dried over Na₂SO₄ and concentrated. The residue waschromatographed over silica gel (elution with 15% EtOAc/hexanes) to givethe product as a colorless solid (1.62 g, 50%). R_(f) 0.41 (40%EtOAc/hexanes). Electrospray Mass Spectrum (50/50 acetonitrile/water)m/z 496 (M−H).

(c)(S)-2-tert-Butoxycarbonylamino-3-(4-ethoxycarbonylmethoxy-3-iodo-phenyl)-propionicacid benzyl ester

To a solution of (S)-2-tertbutoxycarbonylamino-3-(4-hydroxy-3-iodo-phenyl)-propionic acid benzylester (1.88 g, 3.78 mmol) in MeCN (20 mL) was added cesium carbonate(1.85 g, 5.68 mmol) and ethylbromoacetate (0.63 mL, 5.7 mmol). Thereaction was allowed to stir at rt for 64 h. The precipitate was removedby filtration and the filtrate concentrated. The residue waschromatographed over silica gel (elution with a gradient 15-33%EtOAc/hexanes) to give the product as a colorless solid (2.00 g, 91%).R_(f) 0.26 (40% EtOAc/hexanes). Electrospray Mass Spectrum (50/50acetonitrile/water) m/z 606 (M+Na).

(d)3-(3-Allyl-4-ethoxycarbonylmethoxy-phenyl)-(S)-2-tert-butoxycarbonylamino-propionicacid benzyl ester

To a solution of(S)-2-tert-butoxycarbonylamino-3-(4-ethoxycarbonylmethoxy-3-iodo-phenyl)-propionicacid benzyl ester (1.99 g, 3.41 mmol) in DMF (15 mL) was added lithiumchloride (289 mg, 6.82 mmol), allyl tributyltin (1.3 mL, 4.2 mmol) andbis(triphenylphosphine)palladium(II) chloride (143 mg, 0.204 mmol). Themixture was heated (90° C.) and allowed to stir under N₂. The reactionwas allowed to cool to rt and diluted with Et₂O and 0.5 N HCl. Theaqueous phase was extracted with fresh Et₂O. The combined organicextracts were stirred with saturated potassium fluoride for 17 h. Theorganic layer was dried over MgSO₄, concentrated and chromatographed(elution with 30% Et₂O/hexanes). The product was obtained as a colorlesssolid after recrytallization (EtOAc/hexanes) (1.14 g, 67%). R_(f) 0.27(40% Et₂O/hexanes). Electrospray Mass Spectrum (50/50acetonitrile/water) m/z 520 (M+Na).

(e)(S)-2-tert-Butoxycarbonylamino-3-[4-ethoxycarbonylmethoxy-3-(2-oxo-ethyl)-phenyl]-propionicacid benzyl ester

To a solution of3-(3-allyl-4-ethoxycarbonylmethoxy-phenyl)-(S)-2-tert-butoxycarbonylamino-propionicacid benzyl ester (1.00 g, 2.01 mmol) in THF (20 mL) was added pyridine(2 drops) and H₂O (20 mL) followed by osmium tetroxide (1.2 mL, 2.5% wt.in tBuOH, 0.096 mmol). The mixture was allowed to stir for 10 min andNaIO₄ was added portionwise over 0.5 h. The mixture was allowed to stirfor another 0.5 h at rt. The reaction was diluted with H₂O and extractedtwice with Et₂O. The combined organic extracts were washed with brine,dried over MgSO₄ and concentrated. The residue was chromatographed oversilica gel (elution with 25% EtOAc/hexane) to give the product as an oil(902 mg, 90%). R_(f) 0.18 (25% EtOAc/hexanes). Electrospray MassSpectrum (50/50 acetonitrile/water) m/z 522 (M+Na).

(f)(S)-2-tert-Butoxycarbonylamino-3-(4-ethoxycarbonylmethoxy-3-ethoxycarbonylmethyl-phenyl)-propionicacid benzyl ester

To a solution of(S)-2-tert-butoxycarbonylamino-3-[4-ethoxycarbonylmethoxy-3-(2-oxo-ethyl)-phenyl]-propionicacid benzyl ester (880 mg, 1.76 mmol) in tBuOH (35 mL) and cyclohexane(9 mL) was added over 15 min a solution of sodium chlorite (1.79 g, 15.8mmol, 80% tech.) and sodium dihydrogen phosphate dihydrate (1.92 g, 12.3mmol) in H₂O (15 mL). The mixture was allowed to stir at rt for 4 h. Thevolatiles were removed in vacuo and the remaining mixture was acidifiedwith 0.5 N HCl and extracted twice with EtOAc. The combined extractswere washed with brine, dried over Na₂SO₄ and concentrated. The residuewas dissolved in DMF (5 mL) and cesium carbonate (2.87 g, 8.80 mmol) andiodoethane (0.70 mL, 8.7 mmol) were added. The reaction was allowed tostir at rt for 20 h. The reaction was diluted with EtOAc and 0.5 N HCl.The aqueous layer was extracted with fresh EtOAc and the combinedorganic extracts were washed with brine and dried over Na₂SO₄. Thesolution was concentrated and chromatographed over silica gel (elutionwith 25% EtOAc/hexanes) to give the product as a colorless oil (849 mg,89%). R_(f) 0.13 (25% EtOAc/hexanes). Electrospray Mass Spectrum (50/50acetonitrile/water) m/z 567 (M+Na).

(g)(S)-2-tert-Butoxycarbonylamino-3-(4-ethoxycarbonylmethoxy-3-ethoxycarbonylmethyl-phenyl)-propionicacid

To a solution of(S)-2-tert-butoxycarbonylamino-3-(4-ethoxycarbonylmethoxy-3-ethoxycarbonylmethyl-phenyl)-propionicacid benzyl ester (796 mg, 1.46 mmol) in MeOH (8 mL) was added 10% Pd/C(4 mg). The heterogeneous mixture was degassed under reduced pressureand allowed to stir at rt for 18 h under H₂. The catalyst was removed byfiltration and the filtrate evaporated to dryness to give the product asa colorless foam (515 mg, 78%) Electrospray Mass Spectrum (50/50acetonitrile/water) m/z 476 (M+Na).

(h){4-[(S)-2-tert-Butoxycarbonylamino-2-(3-carbamoyl-2-cyclohexylmethoxy-6,7,8,9-tetrahydro-5H-benzocyclohepten-(S)-5-ylcarbamoyl)-ethyl]-2-ethoxycarbonylmethyl-phenoxy}-aceticacid ethyl ester

(S)-2-tert-Butoxycarbonylamino-3-(4-ethoxycarbonylmethoxy-3-ethoxycarbonylmethyl-phenyl)-propionicacid (65 mg, 0.16 mmol) was coupled to(S)-9-amino-3-cyclohexylmethoxy-6,7,8,9-tetrahydro-5H-benzocycloheptene-2-carboxylicacid amide as described for Example X (h). The product was obtained as acolorless solid (92 mg, 78%). R_(f) 0.27 (75% EtOAc/hexane).Electrospray Mass Spectrum (50/50 acetonitrile/water) m/z 774 (M+Na)

(i){4-[(S)-2-Acetylamino-2-(3-carbamoyl-2-cyclohexylmethoxy-6,7,8,9-tetrahydro-5H-benzocyclohepten-(S)-5-ylcarbamoyl)-ethyl]-2-carboxymethyl-phenoxy}-aceticacid

To a cooled (0° C.) solution of{4-[(S)-2-tert-Butoxycarbonylamino-2-(3-carbamoyl-2-cyclohexylmethoxy-6,7,8,9-tetrahydro-5H-benzocyclohepten-(S)-5-ylcarbamoyl)-ethyl]-2-ethoxycarbonylmethyl-phenoxy}-aceticacid ethyl ester (92 mg, 0.12 mmol) in DCM (0.5 mL) was added TFA (0.5mL). After 10 min, the reaction was allowed to warm to rt and after 1 h,the reaction was evaporated to dryness. The crude material was dissolvedin DCM (0.5 mL) and DMA (0.5 mL) and 4-methylmorpholine (16 μL, 0.17mmol) and acetic anhydride (25 μL, 0.23 mmol) were added. The reactionwas allowed to stir at rt for 18 h. The reaction was diluted with 0.5NHCl and extracted twice with EtOAc. The combined extracts were washedwith brine, dried over Na₂SO₄ and concentrated to a colorless oil. Theresidue was dissolved in MeOH (0.7 mL) and DMF (0.7 mL) and cooled (0°C.). To this solution was added lithium hydroxide monohydrate (31 mg,0.74 mmol) in H₂O (0.6 mL). After 0.5 h, the reaction was allowed towarm to rt and after 1.5 h, the reaction was neutralized by carefuladdition of 6 N HCl. Purification by RP-HPLC gave the product as acolorless solid after lyophilization (23 mg, 30%). Electrospray MassSpectrum (50/50 acetonitrile/water) m/z 638 (M+H)

Example 23({4-[(S)-2-Acetylamino-2-(3-carbamoyl-2-cyclohexylmethoxy-6,7,8,9-tetrahydro-5H-benzocyclohepten-(S)-5-ylcarbamoyl)-ethyl]-phenyl}-hydroxyacetyl-amino)-aceticacid

(a){4-[(S)-2-tert-butoxycarbonylamino-2-(3-carbamoyl-2-cyclohexylmethoxy-6,7,8,9-tetrahydro-5H-benzocyclohepten-(S)-5-ylcarbamoyl)-ethyl]-phenyl}-carbamicacid benzyl ester

3-(4-Benzyloxycarbonylamino-phenyl)-(S)-2-tert-butoxycarbonylamino-propionicacid (417 mg, 0.16 mmol) was coupled to(S)-9-amino-3-cyclohexylmethoxy-6,7,8,9-tetrahydro-5H-benzocycloheptene-2-carboxylicacid amide as described for Example X (h). The product was obtained as acolorless solid (231 mg, 65%). R_(f) 0.28 (5% MeOH/CHCl₃). ElectrosprayMass Spectrum (50/50 acetonitrile/water) m/z 713 (M+H).

(b)({4-[(S)-2-Amino-2-(3-carbamoyl-2-cyclohexylmethoxy-6,7,8,9-tetrahydro-5H-benzocyclohepten-(S)-5-ylcarbamoyl)-ethyl]-phenyl}-ethoxyacetyl-amino)-aceticacid ethyl ester

To a solution of{4-[(S)-2-tert-butoxycarbonylamino-2-(3-carbamoyl-2-cyclohexylmethoxy-6,7,8,9-tetrahydro-5H-benzocyclohepten-(S)-5-ylcarbamoyl)-ethyl]-phenyl}-carbamicacid benzyl ester (231 mg, 0.324 mmol) in MeOH (5 mL) was added 10% Pd/C(10 mg). The heterogeneous mixture was degassed under reduced pressureand allowed to stir at rt for 46 h under H₂. The catalyst was removed byfiltration and the filtrate evaporated to dryness. The residue wasdissolved in MeCN (5 mL) and cesium carbonate (264 mg, 0.810 mmol) andethylbromoacetate (0.36 mL, 3.25 mmol) were added. The reaction waswarmed (90° C.) and allowed to stir for 40 h. A second portion ofethylbromoacetate (0.18 mL was added and the reaction allowed to stir at90° C. for 18 h. The reaction was concentrated, diluted with H₂O andextracted twice with EtOAc. The combined organic extracts were washedwith brine, dried over Na₂SO₄ and concentrated. The residue waschromatographed over silica gel (elution with a stepwise gradient 2-4%MeOH/CHCl₃) to give the product as a colorless solid (132 mg, 63%).R_(f) 0.18 (5% MeOH/CHCl₃). Electrospray Mass Spectrum (50/50acetonitrile/water) m/z 651 (M+H).

(c)({4-[(S)-2-Acetylamino-2-(3-carbamoyl-2-cyclohexylmethoxy-6,7,8,9-tetrahydro-5H-benzocyclohepten-(S)-5-ylcarbamoyl)-ethyl]-phenyl}-ethoxyacetyl-amino)-aceticacid ethyl ester

To a solution of({4-[(S)-2-amino-2-(3-carbamoyl-2-cyclohexylmethoxy-6,7,8,9-tetrahydro-5H-benzocyclohepten-(S)-5-ylcarbamoyl)-ethyl]-phenyl}-ethoxyacetyl-amino)-aceticacid ethyl ester (132 mg, 0.203 mmol) in DCM (1.5 mL) was added4-methylmorpholine (27 μL, 0.24 mmol) and acetic anhydride (0.23 μL,0.24 mmol). Allowed to stir at rt for 18 h. The reaction was dilutedwith 0.5 N HCl and extracted twice with EtOAc. The combined extractswere washed with brine, dried over Na₂SO₄ and concentrated. The residuewas chromatographed over silica gel (elution with a stepwise gradient1-3% MeOH/CHCl₃) to give the product as a colorless solid (95 mg, 68%).R_(f) 0.41 (5% MeOH/CHCl₃). Electrospray Mass Spectrum (50/50acetonitrile/water) m/z 693 (M+H).

(d)({4-[(S)-2-Acetylamino-2-(3-carbamoyl-2-cyclohexylmethoxy-6,7,8,9-tetrahydro-5H-benzocyclohepten-(S)-5-ylcarbamoyl)-ethyl]-phenyl}-hydroxyacetyl-amino)-aceticacid

To a solution of({4-[(S)-2-acetylamino-2-(3-carbamoyl-2-cyclohexylmethoxy-6,7,8,9-tetrahydro-5H-benzocyclohepten-(S)-5-ylcarbamoyl)-ethyl]-phenyl}-ethoxyacetyl-amino)-aceticacid ethyl ester (80 mg, 0.12 mmol) in MeOH (1 mL) was added lithiumhydroxide monohydrate (15 mg, 0.36 mmol) in H₂O (1 mL). After 3 h, thereaction was diluted with MeOH (4 mL) and DMF (1 mL) and purified byRP-HPLC (MeCN/H₂O) to give the product as a colorless solid afterlyophilization (25 mg, 34%). Electrospray Mass Spectrum (50/50acetonitrile/water) m/z 635 (M−H).

What is claimed is:
 1. A compound of the formula:

wherein Y is

each occurrence of G is —O—, —S— or —NR—; R⁶ comprises —APO₃RR, —OPO₃RR,—ASO₃R, —OSO₃R, —ACO₂R, —A-tetrazole, —A—N—(PO₃RR)(PO₃RR), —ASO₂NRR,—ACOCF₃, —(C═O)J, —C(R)(J)(K) or —C(Z)(J)(K); where each occurrence of Ais independently a covalent bond, —G—M— or —(M)_(m)—; each occurrence ofM is an independently selected, substituted or unsubstituted, methylenemoiety, and any M-M moiety may be electronically saturated orunsaturated; each n is independently 0, 1, 2, 3, 4 or 5; each m isindependently 0, 1 or 2; J and K are independently —APO₃RR, —OPO₃RR,—ASO₃R, —OSO₃R, —ACO₂R, —A-tetrazole, —ASO₂NRR, —(M)_(n)—NRR or—(M)_(n)—OR; Z is a halogen; R⁷ and R⁸ are independently R, —CN, —NO₂,Z, J, —A(M)_(n)aliphatic, —G(M)_(n)aliphatic, —(M)_(n)COCF₃, —(M)_(n)OH,—(M)_(n)COOR, —A—(M)_(n)NRR, —G(M)_(q)NRR, —(M)_(n)CHO,—A(M)_(n)N(R)(CO)R, —A(M)_(n)N(R)(CO)GR, —G(M)_(q)N(R)(CO)R,—G(M)_(q)N(R)(CO)GR, —A—(M)_(n)—CO—NRR, or —G(M)_(n)—CO—NRR, where thealiphatic groups may be substituted or unsubstituted; or R⁷ is acovalent bond to an R⁴ substituent of X to form an aliphatic, aryl orheterocyclic ring of 4 to 8 atoms; each occurrence of R (unnumbered)represents hydrogen or an aliphatic, heteroaliphatic, aryl, heteroaryl,(aryl)aliphatic-, or (heteroaryl)aliphatic- moiety, each of which (otherthan hydrogen) may be substituted or unsubstituted; q is an integer from1 to 8; X is: —CR³R⁴)_(m)— or —NR⁴—; R³ is hydrogen, R(CO)NR—,RRN(CO)NR—, RSO₂NR—, RCSNR—, RRNCSNR—, RRNSO₂NR—, ROCONR—, RRN—, or

and, R⁴ is hydrogen, aliphatic, cycloaliphatic-(M)_(n)—, aryl-(M)_(n)—,heterocyclic-(M)_(n—, R—SO) ₂M_(n)—, (RO—CO)(M)_(n)— or(RRN—CO)(M)_(n)—, where the aliphatic, cycloaliphatic, aryl orheterocyclic moiety is substituted or unsubstituted; p is 1, 2, 3 or 4;R⁹, R¹⁰, R¹¹ and R¹² are independently —(M)_(n)Z, —(M)_(n)R, —(M)_(n)GR,—(M)_(n)WR, —(M)_(n)WGR, or —(M)_(n)W—COR, or R⁹ and R¹⁰ are covalentlylinked together to form an aliphatic, hetercyclic or aryl fused ring;and, U and W are independently —CO—, —CS—, —M—, —SO—, or —SO₂—; or apharmaceutically acceptable derivative thereof.
 2. A compound of claim 1of the formula:


3. A compound of claim 1 wherein each n is independently 0, 1 or
 2. 4. Acompound of claim 1 wherein X is —CH(NH₂)—.
 5. A compound of claim 1 ofthe formula

where R⁵ comprises a substituted or unsubstituted lower aliphatic oralkoxyl or is a substituted or unsubstituted —(M)_(n)-aryl or—(M)_(n)-heterocyclic group.
 6. A compound of claim 5 wherein R⁵comprises —(M)_(n)CH₃, —(M)_(n)aryl, —(M)_(n)heterocyclic, —(M)_(n)CN,—(M)_(n)COOR, —O—(M)_(n)CH₃, —O(M)_(n)aryl, —O(M)_(n)heterocyclic,—O(M)_(n)CN, or —O(M)_(n)COOR, where n is 0, 1, 2, 3, 4, or
 5. 7. Acompound of claim 5 wherein R⁵ is a substituted or unsubstituted methyl,ethyl, i-propyl, n-propyl, n-butyl, isobutyl, t-butyl, cyclobutyl,n-amyl, sec-amyl, isoamyl, cyclopentyl, n-hexyl, sec-hexyl, isohexyl,cyclohexyl or benzyl.
 8. A compound of claim 5 wherein R⁵ comprises—(CH₂)_(n)CH₃, —(CH₂)(CH₂)_(n)aryl, —(CH₂)(CH₂)_(n)heterocyclic,—(CH₂)(CH₂)_(n)CN, —(CH₂)(CH₂)_(n)COOR, —O(CH₂)_(n)CH₃,—O(CH₂)(CH₂)_(n)aryl, —O(CH₂)(CH₂)_(n)heterocyclic, —O(CH₂)(CH₂)_(n)CN,or —O(CH₂)(CH₂)_(n)COOR, where n is 0, 1, 2, 3, 4, or
 5. 9. A compoundof claim 8 wherein R⁵ comprises —CH₂CN, —CH₂phenyl, —CH₂aryl,—CH₂heterocyclic, —CH₂COOR, —(CH₂)₂COOR, —(CH₂)₃COOR, —(CH₂)₄COOR, whereR is H, lower alkyl or benzyl.
 10. A compound of claim 1 of the formula

wherein R⁵ comprises —O—(M)_(n)CH₃, —O(M)_(n)aryl,—O(M)_(n)heterocyclic, —O(M)_(n)CN, or —O(M)_(n)COOR, where n is 0, 1,2, 3,4, or
 5. 11. A compound of claim 10 wherein R⁵ comprises—O(CH₂)_(n)CH₃, —O(CH₂)(CH₂)_(n)aryl, —O(CH₂)(CH₂)_(n)heterocyclic,—O(CH₂)(CH₂)_(n)CN, or —O(CH₂)(CH₂)_(n)COOR, where n is 0, 1, 2, 3, 4,or
 5. 12. A compound of claim 10 wherein R⁵ comprises —O-(substituted orunsubstituted lower alkyl or benzyl).
 13. A compound of claim 1 of theformula

where R⁴ is hydrogen, substituted or unsubstituted aliphatic (which maybe branched, unbranched or cyclic), substituted or unsubstitutedaryl-(M)_(n)—, substituted or unsubstituted heterocyclic-(M)_(n)—, or(CO₂R)(M)_(n)—.
 14. A compound of claim 13 wherein R⁴ is —(M)_(n)(CO)OR,—(M)_(n)SO₂R, —(M)_(n)(CO)NRR, or —(M)_(n)(tetrazole).
 15. A compound ofclaim 14 wherein R⁴ is —CH₂COOR, —CH₂SO₂R, —CH₂(CO)NRR, or -tetrazole.16. A compound of claim 14 wherein each R is independently H, loweralkyl or benzyl.
 17. A compound of claim 1 of the formula

where R⁴ is hydrogen, substituted or unsubstituted aliphatic (which maybe branched, unbranched or cyclic), substituted or unsubstitutedaryl-(M)_(n)—, substituted or unsubstituted heterocyclic-(M)_(n)—, or(CO₂R)(M)_(n)—.
 18. A compound of claim 17 wherein R⁴ is hydrogen.
 19. Acompound of claim 1 of the formula


20. A compound of any claims 5-12 wherein each R is H.
 21. A compound ofany claims 1-19 wherein M is —CH₂—.
 22. A compound of any claims 1-19wherein Y comprises


23. A compound of claim 22 wherein R⁶ comprises —OR, —APO₃RR, —OPO₃RR,—ASO₃R, —OSO₃R, —ACO₂R, —A-tetrazole, —ASO₂NRR, —ACOCF₃, —C(R)(J)(K) or—C(Z)(J)(K); and R⁷ and R⁸ are independently H, —CN, —NO₂, halogen, J,—A—(M)_(n)aliphatic, —G—(M)_(n)aliphatic, —(M)_(n)COCF₃, —(M)_(n)OH,—(M)_(n)COOR, —A—(M)_(n)NRR, —G—(M)_(q)NRR, —(M)_(n)CHO,—A—(M)_(n)N(R)(CO)R, —G—(M)_(q)N(R)(CO)R, —A—(M)_(n)—CO—NRR, or—G—(M)_(n)—CO—NRR, where the aliphatic groups may be substituted orunsubstituted; or R⁷ is a covalent bond to an R⁴ substituent of X toform a ring of 4 to 8 atoms.
 24. A compound of claim 22 wherein R⁶comprises —OR, —APO₃RR, —OPO₃RR, —ACO₂R, —ACOCF₃ or —C(R)(J)(K); Acomprises —CH₂—, —OCH₂—, —CF₂—, —CHF—, —CHOH— or a covalent bond; each RH, or substituted or unsubstituted lower alkyl or substituted orunsubstituted benzyl; and, R⁷ and R⁸ are independently H, J, —A—(M)substituted or unsubstituted aliphatic, —G—(M) substituted orunsubstituted aliphatic, —(M)_(n)COCF₃, —(M)_(n)OH, —(M)_(n)COOR,—A—(M)_(n)NRR, —(M)_(n)CHO, —A—(M)_(n)N(R)(CO)R or —A—(M)_(n)—CO—NRR.25. A compound of claim 22 wherein R⁶ comprises —OH, —PO₃RR, —OPO₃RR,—CH₂PO₃RR, —CF₂PO₃RR, —OCH₂CO₂R, —NHCH₂CO₂R, —CH₂CO₂R, —CF₂CO₂R,—CH₂SO₃R, —CF₂SO₃R, —CH₂COCF₃, —CF₂COCF₃, —CH(PO₃RR)₂, —CH(OH)(PO₃RR),—CH(NH₂)(PO₃RR), —CH(CO₂R)₂, —CF(CO₂R)₂, —CH(PO₃RR)(CO₂R″),—CH(PO₃RR)(SO₃R″), —CH(PO₃RR)(SO₂NH₂), —CH(SO₂NH₂)₂, or —CH(SO₃RR)₂. 26.A compound of claim 25 in which one or more R group in the —PO₃RR,—OPO₃RR, —CH₂PO₃RR, —CF₂PO₃RR, —OCH₂CO₂R, —NHCH₂CO₂R, —CH₂CO₂R,—CF₂CO₂R, —CH₂SO₃R, —CH₂COCF₃, —CF₂COCF₃, —CH(PO₃RR)₂, —CH(OH)(PO₃RR),—CH(NH₂)(PO₃RR), —CH(CO₂R)₂, —CF(CO₂R)₂, —CH(PO₃RR)(CO₂R),—CH(PO₃RR)(SO₃R), —CH(PO₃RR)(SO₂NH₂), —CH(SO₂NH₂)₂, or —CH(SO₃RR)₂moiety is H.
 27. A compound of claim 25 in which one or more R in the—PO₃RR, —OPO₃RR, —CH₂PO₃RR, —CF₂PO₃RR, —OCH₂CO₂R, —NHCH₂CO₂R, —CH₂CO₂R,—CF₂CO₂R, —CH₂SO₃R, —CF₂SO₃R, —CH₂COCF₃, —CF₂COCF₃, —CH(PO₃RR)₂,—CH(OH)(PO₃RR), —CH(NH₂)(PO₃RR), —CH(CO₂R)₂, —CH(PO₃RR)(CO₂R),—CH(PO₃RR)(SO₃R), —CH(PO₃RR)(SO₂NH₂), —CH(SO₂NH₂)₂, or —CH(SO₂RR)₂moiety is —(M)_(m)CH₂Z, —(M)_(m)CHZ₂, —(M)_(m)CZ₃, —R¹⁵, —M—O—CO—R¹⁵ or—M—O—CO—OR⁵, where Z is halogen and R¹⁵ is substituted or unsubstitutedlower aliphatic, aryl or heterocyclic.
 28. A compound of claim 27 inwhich R¹⁵ is methyl, ethyl, n-propyl, i-propyl, n-butyl, isobutyl,t-butyl, n-amyl, sec-amyl, benzyl or substituted benzyl.
 29. A compoundof claim 25 wherein R⁷ and R⁸ are H.
 30. A compound of claim 25 whereinR⁷ is J, —A—(M)_(n)(substituted o aliphatic, aryl or heterocyclic),—G—(M)_(n)(substituted or unsubstituted aliphatic, aryl orheterocyclic), —(M)_(n)COCF₃, —(M)_(n)OH, —(M)_(n)COOR, —A—(M)_(n)NRR,—(M)_(n)CHO, —A—(M)_(n)N(R)(CO)R, —A—(M)_(n)—NRR or —A—(M)_(n)—CO—NRR;and R⁸ is H.
 31. A compound of claim 25 wherein R⁷ is lower alkyl, loweralkenyl, —OH, —NH₂, —NO₂, —CN, —NHR, —NHCOR, —CHO, —CH₂CHO, —PO₃RR,—OPO₃RR, —CH₂PO₃RR, —CF₂PO₃RR, —OCH₂CO₂R, —NHCH₂CO₂R, —CH₂CO₂R,—CF₂CO₂R, —SO₃R, —CH₂SO₃R, —CF₂SO₃R, —COCF₃, —COCH₂F, —COCF₂H, —CF₂COCF₃or —SO₂NH₂.
 32. A compound of claim 31 in which at least one R group in—PO₃RR, —OPO₃RR, —CH₂PO₃RR, —CF₂PO₃RR, —OCH₂CO₂R, —NHCH₂CO₂R, —CH₂CO₂R,—CF₂CO₂R, —SO₃R, —CH₂SO₃R, or —CF₂SO₃R is H.
 33. A compound of claim 31in which at least one R group in —PO₃RR, —OPO₃RR, —CH₂PO₃RR, —CF₂PO₃RR,—OCH₂CO₂R, —NHCH₂CO₂R, —CH₂CO₂R, —CF₂CO₂R, —SO₃R, —CH₂SO₃R, or —CF₂SO₃Ris —(M)_(m)—CH₂Z, —(M)_(m)—CHZ₂, —(M)_(m)—CZ₃, —R¹⁵, —M—O—CO—R¹⁵ or—M—O—CO—OR¹⁵, where Z is halogen and R¹⁵ is substituted or unsubstitutedlower aliphatic, aryl or heterocyclic.
 34. A compound of claim 33 inwhich R¹⁵ is methyl, ethyl, n-propyl, i-propyl, n-butyl, isobutyl,t-butyl, n-amyl, sec-amyl, benzyl or substituted benzyl.
 35. A compoundof claim 29 wherein R⁶ comprises —APO₃RR or —OPO₃RR and R⁷ is—A—(M)_(n)-aliphatic or —G—(M)_(n)-aliphatic, where the aliphatic moietyis substituted or unsubstituted.
 36. A compound of claim 35 wherein R⁶comprises —OPO₃H₂.
 37. A compound of claim 22 wherein R⁶ and R⁷ areindependently selected from J and K.
 38. A compound of claim 22 whereinR⁶ is —C(R)(J)(K).
 39. A compound of claim 38 wherein R is H.
 40. Acompound of claim 38 wherein J is —PO₃RR.
 41. A compound of claim 40 inwhich one or both R groups is H.
 42. A compound of claim 40 in which oneor both R groups is R¹⁵, —(M)_(m)—CH₂Z, —(M)_(m)—CHZ₂, —(M)_(m)—CZ₃,—M—O—CO—R¹⁵ or —M—O—CO—OR¹⁵, where Z is halogen and R¹⁵ is substitutedor unsubstituted lower aliphatic, aryl or heterocyclic.
 43. A compoundof claim 42 in which R¹⁵ is methyl, ethyl, n-propyl, i-propyl, n-butyl,isobutyl, t-butyl, n-amyl, sec-amyl, benzyl or substituted benzyl.
 44. Acompound of claim wherein each of R⁹, R¹⁰, R¹¹ and R¹² is independently—(M)_(n)Z, —(M)_(n)R, —G(M)_(n)R, —(M)_(n)WR or —(M)_(n)W—GR.
 45. Acompound of claim 44 wherein one or more of the R groups contain atleast one substituent selected from halo, hydroxy, or a substituted orunsubstituted aliphatic, amino, amido or sulfonamido moiety.
 46. Acompound of claim 44 wherein one or more of R⁹, R¹⁰, R¹¹, and R¹² is asubstituted aliphatic moiety containing at least one substituentselected from substituted or unsubstituted cycloaliphatic, substitutedor unsubstituted aryl, substituted or unsubstituted heteroaryl, —CO₂R,—CO—NRR, and —OR.
 47. A compound of claim 44, wherein one or more of R⁹,R¹⁰, R¹¹, and R¹² comprises —(M)_(n)(cycloaliphatic),—(M)_(n)(substituted or unsubstituted aryl), —(M)_(n)(substituted orunsubstituted heteroaryl), —(M)_(n)CHO, —(M)_(n)CONH₂, —(M)_(n)CSNH₂,—(M)_(n)SONH₂, —(M)_(n)SO₂NRR, —(M)_(n)OR, —(M)_(n)(lower aliphatic),—(M)_(n)—C(OR)RR, or —(M)_(n)—C═CRR.
 48. A compound of claim 44, whereinone or more of R⁹, R¹⁰, R¹¹, and R¹² comprises —G(M)_(n)(aliphatic),—G(M)_(n)(cycloaliphatic), —G(M)_(n)(substituted or unsubstituted aryl),—G(M)_(n)(substituted or unsubstituted heteroaryl), —G(M)_(n)CHO,—G(M)_(n)CONH₂, —G(M)_(n)CSNH₂, —G(M)_(n)SONH₂, —G(M)_(n)SO₂NRR,—G(M)_(n)OR, —G(M)_(n)(lower aliphatic), —G(M)_(n)—C(OR)RR, or—G(M)_(n)—C═CRR.
 49. A compound of claim 48 wherein —G(M)_(n) comprises—OCH₂—, —SCH₂— or —NRCH₂—.
 50. A compound of claim 44, wherein one ormore of R⁹, R¹⁰, R¹¹, and R¹² comprises methyl, —(CH₂)_(q)R¹³ where q is1-7 and R¹³ comprises methyl; i-propyl; i-butyl; t-butyl;cycloaliphatic; phenyl; substituted phenyl; naphthyl; substitutednaphthyl; a 5, 6 or 7-membered heterocyclic ring or a bicyclicheterocylic moiety.
 51. A compound of claim 44, wherein one or more ofR⁹, R¹⁰, R¹¹, and R¹² comprises —(M)_(n)W—NH—R and one or more of R⁹,R¹⁰, R¹¹, and R¹² comprises —O(M)_(m)(aliphatic).
 52. A compound ofclaim 44 of the formula:


53. A compound of claim 44, wherein one or more of R⁹, R¹⁰, R¹¹, and R¹²comprises —(M)_(n)(CO)—NH—R.
 54. A compound of claim 44, wherein one ormore of R⁹, R¹⁰, R¹¹, and R¹² comprises —CONH—R.
 55. A compound of claim44, wherein one or more of R⁹, R¹⁰, R¹¹, and R¹² comprises—(CH₂)_(m)CONH—R.
 56. A compound of claim 1 of the formula:

where R⁵ comprises a substituted or unsubstituted lower aliphaticmoiety, and R⁹ comprises —(M)_(n)W—NH—R and R¹⁰ comprises—O(M)_(m)(aliphatic).
 57. A compound of claim 56, wherein R⁹ comprises—CONH—R and R¹⁰ comprises —OM-cycloaliphatic or —OM-branched chainaliphatic.
 58. A compound of claim 56, wherein R⁹ comprises —CH₂CONH—Rand R¹⁰ comprises —OM-cycloaliphatic or —OM-branched chain aliphatic.59. A compound of claim 44, wherein Y comprises:

R⁶ comprises —APO₃RR, —ASO₃R, —ACO₂R, —ASO₂NRR, —ACOCF₃, or —C(R)(J)(K);and, R⁷ is H, —CN, —NO₂, halogen, J, —A—(M)_(n)substituted orunsubstituted aliphatic, —(M)_(n)COCF₃, —(M)_(n)OH, —(M)_(n)COOR,—A—(M)_(n)NRR, —(M)_(n)CHO, —A—(M)_(n)N(R)(CO)R or —A—(M)_(n)—CO—NRR.60. A compound of claim 59, wherein R⁶ comprises —OPO(OH)₂, —PO(OH)₂,—OCH₂COOH, —CF₂PO(OH)₂, or —CH(PO₃H₂).
 61. A compound of claim 44,wherein R⁷ comprises H.
 62. A compound of claim 44, wherein R⁷ comprisesCHO.
 63. A compound of claim 44, wherein R⁷ comprises J.
 64. A compoundof claim 44, wherein R⁷ comprises —A—(M)_(n)substituted or unsubstitutedaliphatic.
 65. A compound of claim 44, wherein R⁶ comprises —OPO(OH)₂and R⁷ is H.
 66. A compound of claim 64, wherein (M)_(n) is (CH₂)_(n).67. A compound of claim 66, wherein n is
 1. 68. A compound of claim 59,wherein one or more (unnumbered) R groups comprise —(M)_(m)CH₂Z,—(M)_(m)CHZ₂, —(M)_(m)CZ₃, —M—O—CO—R¹⁵ or —M—O—CO—OR¹⁵, where Z ishalogen and R¹⁵ is a substituted or unsubstituted lower aliphatic, arylor heterocyclic group.
 69. A compound of any of claims 1-19, which bindsto a given SH2 domain with a IC₅₀ value of less than about 50 μM.
 70. Acompound of claim 69, which binds to a given SH2 domain with a IC₅₀value of less than about 20 μM.
 71. A compound of claim 69 wherein theSH2 domain is from a Src, Fyn, Lck, Yes, Blk, Lyn, Fgr, Hck, Yrk,ZAP-70, Syk, STAT or Abl protein.
 72. A composition comprising acompound of any of claims 1-19, or a pharmaceutically acceptablederivative thereof, and a pharmaceutically acceptable excipient.
 73. Amethod for inhibiting SH2-mediated signal transduction in a mammal inneed thereof which comprises administering to the mammal apharmaceutical composition of claim
 72. 74. The method of claim 73,wherein the pharmaceutical composition contains a compound whichspecifically binds to an SH2 domain of Src, ZAP-70, Syk, or STAT
 6. 75.The method of claim 73, wherein said SH2-mediated signal transduction ismediated by a PDGF receptor protein, EGF receptor protein, HER2/Neureceptor protein, fibroblast growth factor receptor protein, focaladhesion kinase protein, p130 protein, or p68 protein.
 76. The method ofclaim 73, wherein the mammal has a proliferative disease, restenosis,osteoporosis, inflammation, allergies, or cardiovascular disease. 77.The method of claim 73, wherein the mammal has a cancer.
 78. A method oftreating a patient who has a proliferative disease, restenosis,osteoporosis, inflammation, allergic reaction, or cardiovasculardisease, the method comprising administering to the patient atherapeutically effective amount of a composition of claim
 72. 79. Amethod of treating a patient who has a cancer, the method comprisingadministering to the patient a therapeutically effective amount of acomposition of claim
 72. 80. A method for inducing immunosuppression ina patient, the method comprising administering to the patient an amountof a composition of claim 72 sufficient to cause immunosuppression. 81.A compound of the structure:

wherein each of R⁹ and R¹⁰ is independently halo, R, —OR, —SR, —NRR,—COR, or —(M)_(n)W—NHR; each occurrence of M is an independentlyselected, substituted or unsubstituted, methylene moiety; each n isindependently 0, 1, 2, 3, 4 or 5; each occurrence of R (unnumbered)represents hydrogen or an aliphatic, heteroaliphatic, aryl, heteroaryl,(aryl)aliphatic-, or (heteroaryl)aliphatic-moiety, each of which (otherthan hydrogen) may be substituted or unsubstituted; and, W is —CO—,—CS—, —M—, —SO—, or —SO₂—.